Dioxino- and oxazin-[2,3-D]pyrimidine PI3K inhibitor compounds and methods of use

ABSTRACT

Dioxino- and oxazin-[2,3-d]pyrimidine PI3K inhibitor compounds of Formula I with anti-cancer activity, anti-inflammatory activity, or immunoregulatory properties, and more specifically with PI3 kinase modulating or inhibitory activity are described. Methods are described for using the tricyclic PI3K inhibitor compounds of Formula I for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, organisms, or associated pathological conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed under 37 CFR §1.53(b), claims thebenefit under 35 USC §119(e) of U.S. Provisional Application Ser. No.61/694,898 filed on 30 Aug. 2012, which is incorporated by reference inentirety.

FIELD OF THE INVENTION

The invention relates generally to compounds with anti-cancer activityor anti-inflammatory activity, and more specifically to compounds whichinhibit PI3 kinase activity. The invention also relates to methods ofusing the compounds for in vitro, in situ, and in vivo diagnosis ortreatment of mammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Phosphatidylinositol is one of a number of phospholipids found in cellmembranes which play an important role in intracellular signaltransduction. Cell signaling via 3′-phosphorylated phosphoinositides hasbeen implicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity(Rameh et al (1999) J. Biol Chem, 274:8347-8350). The enzyme responsiblefor generating these phosphorylated signaling products,phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K),was originally identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylate phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al(1992) Trends Cell Biol 2:358-60).

Phosphoinositide 3-kinases (PI3K) are lipid kinases that phosphorylatelipids at the 3-hydroxyl residue of an inositol ring (Whitman et al(1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP3s)generated by PI3-kinases act as second messengers recruiting kinaseswith lipid binding domains (including plekstrin homology (PH) regions),such as Akt and phosphoinositide-dependent kinase-1 (PDK1). Binding ofAkt to membrane PIP3s causes the translocation of Akt to the plasmamembrane, bringing Akt into contact with PDK1, which is responsible foractivating Akt. The tumor-suppressor phosphatase, PTEN, dephosphorylatesPIP3 and therefore acts as a negative regulator of Akt activation. ThePI3-kinases Akt and PDK1 are important in the regulation of manycellular processes including cell cycle regulation, proliferation,survival, apoptosis and motility and are significant components of themolecular mechanisms of diseases such as cancer, diabetes and immuneinflammation (Vivanco et al (2002) Nature Rev. Cancer 2:489; Phillips etal (1998) Cancer 83:41).

The main PI3-kinase isoform in cancer is the Class I PI3-kinase, p110α(alpha) (U.S. Pat. No. 5,824,492; U.S. Pat. No. 5,846,824; U.S. Pat. No.6,274,327). Other isoforms are implicated in cardiovascular andimmune-inflammatory disease (Workman P (2004) Biochem Soc Trans32:393-396; Patel et al (2004) Proceedings of the American Associationof Cancer Research (Abstract LB-247) 95th Annual Meeting, March 27-31,Orlando, Fla., USA; Ahmadi K and Waterfield Md. (2004) Encyclopedia ofBiological Chemistry (Lennarz W J, Lane M D eds) Elsevier/AcademicPress). The PI3 kinase/Akt/PTEN pathway is an attractive target forcancer drug development since such modulating or inhibitory agents wouldbe expected to inhibit proliferation, reverse the repression ofapoptosis and surmount resistance to cytotoxic agents in cancer cells(Folkes et al (2008) J. Med. Chem. 51:5522-5532; Yaguchi et al (2006)Jour. of the Nat. Cancer Inst. 98(8):545-556).

Malignant gliomas are the most common primary brain tumors in adults. Inglioblastoma (GBM), the most aggressive glioma subtype, tumor formationand growth appear to be driven by amplification or overexpression ofgene products involved in growth factor-initiated signal transductionacting in cooperation with genetic alterations disrupting cell-cyclecontrol (Holland EC (2001) Nat Rev Genet 2:120-129). Of the genomicalterations described in GBM, PTEN mutation and/or deletion is the mostcommon, with an estimated frequency of 70-90% (Nutt C, Louis D N (2005)Cancer of the Nervous System (McGraw-Hill, New York), 2nd Ed, pp837-847.). These findings, along with the prognostic value of PTENstatus in GBM cases (Phillips H S, et al. (2006) Cancer Cell 9:157-163),suggest the importance of the phosphoinositide 3-kinase (PI3K)/Aktpathway in promoting highly aggressive glial malignancies, as well asthe opportunities for treatment with PI3K inhibitors possessingblood-brain barrier penetrant properties.

Malignant gliomas are treated with a combination of surgery, radiation,and temozolomide (TEMODAR™), but these therapies ultimately fail at ahigh frequency due to tumor recurrence. Additional therapies are neededfor delivery of effective concentrations of effective drugs to the brainto treat hyperproliferative disorders, such as glioblastoma andmetastatic brain cancer.

SUMMARY OF THE INVENTION

The invention relates generally to dioxino-oxazin-[2,3-d]pyrimidine PI3Kinhibitor compounds of Formula I with anti-cancer activity,anti-inflammatory activity, or immunoregulatory properties, and morespecifically with PI3 kinase modulating or inhibitory activity. Certainhyperproliferative disorders are characterized by the modulation of PI3kinase function, for example by mutations or overexpression of theproteins. Accordingly, the compounds of the invention may be useful inthe treatment of hyperproliferative disorders such as cancer. Thecompounds may inhibit tumor growth in mammals and may be useful fortreating human cancer patients.

The invention also relates to methods of using thedioxino-oxazin-[2,3-d]pyrimidine PI3K inhibitor compounds of Formula Ifor in vitro, in situ, and in vivo diagnosis or treatment of mammaliancells, organisms, or associated pathological conditions.

Formula I compounds include:

and stereoisomers, geometric isomers, tautomers, and pharmaceuticallyacceptable salts thereof. The substituents are as described herein.

Another aspect of the invention provides a pharmaceutical compositioncomprising a dioxino[2,3-d]pyrimidine PI3K inhibitor compound of FormulaI and a pharmaceutically acceptable carrier. The pharmaceuticalcomposition may further comprise one or more additional therapeuticagent.

Another aspect of the invention provides methods of inhibiting PI3kinase activity, comprising contacting a PI3 kinase with an effectiveinhibitory amount of a compound of Formula I.

Another aspect of the invention provides methods of preventing ortreating a hyperproliferative disease or disorder modulated by PI3kinases, comprising administering to a mammal in need of such treatmentan effective amount of a compound of Formula I. Examples of suchhyperproliferative disease or disorder include, but are not limited to,cancer.

Another aspect of the invention provides methods of preventing ortreating a hyperproliferative disorder, comprising administering to amammal in need of such treatment an effective amount of a compound ofFormula I, alone or in combination with one or more additional compoundshaving anti-hyperproliferative properties.

In a further aspect the present invention provides a method of using acompound of this invention to treat a hyperproliferative disease orcondition modulated by PI3 kinase in a mammal.

An additional aspect of the invention is the use of a compound of thisinvention for treating cancer modulated by PI3 kinase in a mammal,including brain cancer.

Another aspect of the invention includes kits comprising a compound ofFormula I, a container, and optionally a package insert or labelindicating a treatment.

Another aspect of the invention includes methods of preparing, methodsof separating, and methods of purifying compounds of Formula I.

Another aspect of the invention includes novel intermediates useful forpreparing Formula I compounds.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.

Definitions

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂)_(,) 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, e.g., as a bicyclic[4,5], [5,5],[5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10 ringatoms can be arranged as a bicyclic[5,6] or [6,6] system, or as bridgedsystems such as bicyclic[2.2.1]heptane, bicyclic[2.2.2]octane andbicyclic[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), e.g.:bicyclic[4,5], [5,5], [5,6], or [6,6] system. Heterocycles are describedin Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and9; “The Chemistry of Heterocyclic Compounds, A series of Monographs”(John Wiley & Sons, New York, 1950 to present), in particular Volumes13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.“Heterocyclyl” also includes radicals where heterocycle radicals arefused with a saturated, partially unsaturated ring, or aromaticcarbocyclic or heterocyclic ring. Examples of heterocyclic ringsinclude, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, e.g., 2-hydroxypyridinyl),imidazolyl, imidazopyridinyl, pyrimidinyl (including, e.g.,4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl,tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl,thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionallysubstituted independently with one or more substituents describedherein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats or prevents theparticular disease, condition, or disorder, (ii) attenuates,ameliorates, or eliminates one or more symptoms of the particulardisease, condition, or disorder, or (iii) prevents or delays the onsetof one or more symptoms of the particular disease, condition, ordisorder described herein. In the case of cancer, the therapeuticallyeffective amount of the drug may reduce the number of cancer cells;reduce the tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the cancer. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy can be measured, e.g., byassessing the time to disease progression (TTP) and/or determining theresponse rate (RR).

The terms “cancer” refers to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth. A“tumor” comprises one or more cancerous cells. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine,dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CASNo. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), pemetrexed (ALIMTA®, Eli Lilly), trastuzumab(HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNEO, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Il),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes, verracurin A, roridin Aand anguidine); urethan; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”);cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine(NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, e.g., tamoxifen (including NOLVADEX®; tamoxifencitrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate);(ii) aromatase inhibitors that inhibit the enzyme aromatase, whichregulates estrogen production in the adrenal glands, such as, e.g.,4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate),AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR®(vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole;AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); (iv) protein kinaseinhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinaseinhibitors; (vi) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation, e.g., PKC-alpha, Raf and H-Ras, such as oblimersen(GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expressioninhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii)vaccines such as gene therapy vaccines, e.g., ALLOVECTIN®, LEUVECTIN®,and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such asLURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the PI3K inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result e.g. from the oxidation, reduction, hydrolysis,amidation, deamidation, esterification, deesterification, enzymaticcleavage, and the like, of the administered compound. Accordingly, theinvention includes metabolites of compounds of the invention, includingcompounds produced by a process comprising contacting a compound of thisinvention with a mammal for a period of time sufficient to yield ametabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, e.g., treatment of the free base with an inorganic acid, such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, trifluoroacetic acid, maleic acid, succinicacid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalicacid, glycolic acid, salicylic acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha-hydroxy acid, such ascitric acid or tartaric acid, an amino acid, such as aspartic acid orglutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid,a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid,or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,e.g., treatment of the free acid with an inorganic or organic base, suchas an amine (primary, secondary or tertiary), an alkali metal hydroxideor alkaline earth metal hydroxide, or the like. Illustrative examples ofsuitable salts include, but are not limited to, organic salts derivedfrom amino acids, such as glycine and arginine, ammonia, primary,secondary, and tertiary amines, and cyclic amines, such as piperidine,morpholine and piperazine, and inorganic salts derived from sodium,calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminumand lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The terms “compound of this invention,” and “compounds of the presentinvention” and “compounds of Formula I” include compounds of Formulas Iand stereoisomers, geometric isomers, tautomers, solvates, metabolites,and pharmaceutically acceptable salts and prodrugs thereof.

Any formula or structure given herein, including Formula I compounds, isalso in intended to represent hydrates, solvates, and polymorphs of suchcompounds, and mixtures thereof.

Dioxino- and Oxazin-[2,3-D]Pyrimidine PI3K Inhibitor Compounds

The present invention provides dioxino- and oxazin-[2,3-d]pyrimidinePI3K inhibitor compounds of Formula I, and pharmaceutical formulationsthereof, which are potentially useful in the treatment of diseases,conditions and/or disorders modulated by PI3 kinases (PI3K). Morespecifically, the present invention provides compounds of Formula I:

and stereoisomers, geometric isomers, tautomers, and pharmaceuticallyacceptable salts thereof, wherein:

R¹ and R² are independently selected from H, ═O, C₁-C₆ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, and C₃-C₁₂ carbocyclyl, where alkyl andcarbocyclyl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃, —CH(CH₃)₂,—CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN, —CF₃, —CH₂OH, —CO₂H, —CONH₂,—CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —OH, ═O,—OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(═O)NHCH₃, —NHC(═O)NHCH₂CH₃,—NHC(═O)NHCH(CH₃)₂, —NHS(O)₂CH₃, —N(CH₃)C(═O)OC(CH₃)₃, —S(O)₂CH₃,benzyl, benzyloxy, cyclopropyl, morpholinyl, morpholinomethyl, and4-methylpiperazin-1-yl; or

-   -   the R¹ groups or the R² groups form a spiro, 3- to 6-membered        carbocyclic or heterocyclic ring;    -   R³ is selected from C₆-C₂₀ aryl, C₂-C₂₀ heterocyclyl and C₁-C₂₀        heteroaryl, each of which are optionally substituted with one or        more groups independently selected from F, Cl, Br, I, —CH₃,        —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN, —CF₃,        —CH₂OH, —CO₂H, —CONH₂, —CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂,        —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂,        —SH, —NHC(═O)NHCH₃, —NHC(═O)NHCH₂CH₃, —NHC(═O)NHCH(CH₃)₂,        —NHS(O)₂CH₃, —N(CH₃)C(═O)OC(CH₃)₃, —S(O)₂CH₃, benzyl, benzyloxy,        morpholinyl, morpholinomethyl, and 4-methylpiperazin-1-yl;

X is selected from O, S, and NR;

R is H or C₁-C₆ alkyl.

Further it is to be understood that every embodiment relating to aspecific residue R¹, R², R³, as disclosed herein may be combined withany other embodiment relating to another residue R¹, R², R³, asdisclosed herein.

Exemplary embodiments of compounds of the invention include Formulas Iaand Ib:

Exemplary embodiments of Formula I compounds include wherein R¹ isindependently selected from H, —CH₃, —CH₂CH₃, —C(CH₃)₃, —CH₂OH,—CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃, —CN, —CH₂F, —CHF₂, and —CF₃.

Exemplary embodiments of Formula I compounds include wherein R¹ isindependently selected from H, —CH₃, and cyclopropyl.

Exemplary embodiments of Formula I compounds include wherein R² isindependently selected from H, —CH₃, —C(CH₃)₃, —CH₂OH, —CF₃, —CH₂F, andcyclopropyl.

Exemplary embodiments of Formula I compounds include wherein R² are each—CH₃.

Exemplary embodiments of Formula I compounds include wherein two R² form═O or cyclopropyl.

Exemplary Formula I compounds include wherein R³ is phenyl substitutedwith one or more groups selected from F, Cl, Br, I, —CH₃, —CH₂CH₃,—CH(CH₃)₂, —CN, —CF₃, —CH₂OH, —CO₂H, —CONH₂, —CONH(CH₃), —CON(CH₃)₂,—NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH(CH₃)₂, —SH, —NHC(═O)NHCH₃, —NHC(═O)NHCH₂CH₃, —NHS(O)₂CH₃,—N(CH₃)C(═O)OC(CH₃)₃, and —S(O)₂CH₃.

Exemplary Formula I compounds include wherein R³ is an optionallysubstituted bicyclic heteroaryl group selected from 1H-indazole,1H-indole, indolin-2-one, 1-(indolin-1-yl)ethanone,1H-benzo[d][1,2,3]triazole, 1H-pyrazolo[3,4-b]pyridine,1H-pyrazolo[3,4-d]pyrimidine, 1H-benzo[d]imidazole,1H-benzo[d]imidazol-2(3H)-one, 1H-pyrazolo[3,4-c]pyridine,1H-pyrrolo[2,3-c]pyridine, 3H-imidazo[4,5-c]pyridine,7H-pyrrolo[2,3-d]pyrimidine, 7H-purine, 1H-pyrazolo[4,3-d]pyrimidine,5H-pyrrolo[3,2-d]pyrimidine, 2-amino-1H-purin-6(9H)-one, quinoline,quinazoline, quinoxaline, isoquinoline, isoquinolin-1(2H)-one,3,4-dihydroisoquinolin-1(2H)-one, 3,4-dihydroquinolin-2(1H)-one,quinazolin-2(1H)-one, quinoxalin-2(1H)-one, 1,8-naphthyridine,pyrido[3,4-d]pyrimidine, and pyrido[3,2-b]pyrazine.

Exemplary Formula I compounds include wherein optionally substituted R³is selected from:

where the wavy line indicates the site of attachment.

In one embodiment of the invention R³ is 1H-indazol-4-yl.

Exemplary Formula I compounds include wherein R³ is an optionallysubstituted monocyclic heteroaryl group selected from 2-furanyl,3-furanyl, 2-imidazolyl, 4-imidazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-pyrazolyl,4-pyrazolyl, 2-pyrazinyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl,2-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 5-tetrazolyl,1-tetrazolyl, 2-tetrazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,3-triazolyl, and 1-triazolyl.

Exemplary Formula I compounds include optionally substituted R³ isselected from:

where the wavy line indicates the site of attachment.

Exemplary Formula I compounds include wherein R³ is an optionallysubstituted monocyclic heteroaryl group selected from pyridyl,pyrimidinyl or pyrazolyl.

Exemplary Formula I compounds include wherein R³ is an optionallysubstituted pyrimidinyl.

In one embodiment of the invention R³ is 2-aminopyrimidin-5-yl.

Exemplary Formula I compounds include optionally substituted R³:

where the wavy line indicates the site of attachment.

The Formula I compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention.

In addition, the present invention embraces all geometric and positionalisomers. For example, if a Formula I compound incorporates a double bondor a fused ring, the cis- and trans-forms, as well as mixtures thereof,are embraced within the scope of the invention. Both the singlepositional isomers and mixture of positional isomers are also within thescope of the present invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

The present invention also embraces isotopically-labeled compounds ofthe present invention which are identical to those recited herein, butfor the fact that one or more atoms are replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. All isotopes of any particular atom or elementas specified are contemplated within the scope of the compounds of theinvention, and their uses. Exemplary isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine,such as ²H (D), ³H, ¹¹C, ¹³C, ¹³N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, ³³P, ³⁵S, ¹⁸F,³⁶Cl, ¹²³I and ¹²⁵I. Certain isotopically-labeled compounds of thepresent invention (e.g., those labeled with ³H and ¹⁴C) are useful incompound and/or substrate tissue distribution assays. Tritiated (³H) andcarbon-14 (¹⁴C) isotopes are useful for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸Fare useful for positron emission tomography (PET) studies to examinesubstrate receptor occupancy. Isotopically labeled compounds of thepresent invention can generally be prepared by following proceduresanalogous to those disclosed in the Schemes and/or in the Examplesherein below, by substituting an isotopically labeled reagent for anon-isotopically labeled reagent.

Biological Evaluation

Determination of the PI3 kinase activity of a Formula I compound ispossible by a number of direct and indirect detection methods. Certainexemplary compounds described herein were assayed for their p110α(alpha), and other isoform, PI3K binding activity (Example 901) and invitro activity against tumor cells (Example 902). Certain exemplarycompounds of the invention had PI3K binding activity IC₅₀ values lessthan 10 nM. Certain compounds of the invention had tumor cell-basedactivity EC₅₀ values less than 100 nM.

The cytotoxic or cytostatic activity of Formula I exemplary compoundswas measured by: establishing a proliferating mammalian tumor cell linein a cell culture medium, adding a Formula I compound, culturing thecells for a period from about 6 hours to about 5 days; and measuringcell viability (Example 902). Cell-based in vitro assays were used tomeasure viability, i.e. proliferation (IC₅₀), cytotoxicity (EC₅₀), andinduction of apoptosis (caspase activation).

The in vitro potency of Formula I exemplary compounds was measured bythe cell proliferation assay, CellTiter-Glo® Luminescent Cell ViabilityAssay, commercially available from Promega Corp., Madison, Wis. (Example902). This homogeneous assay method is based on the recombinantexpression of Coleoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat.No. 5,674,713; U.S. Pat. No. 5,700,670) and determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al (1993) J. Immunol.Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 or 384 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative effects of Formula I exemplary compounds weremeasured by the CellTiter-Glo® Assay (Example 902) against several tumorcell lines. Potency EC₅₀ values were established for the testedcompounds. The range of in vitro cell potency activities was about 100nM to about 10 μM. Certain tested compounds had EC₅₀ values of less than1 micromolar (1 μM) in stopping proliferation of certain tumor celllines.

Certain ADME properties were measured for certain exemplary compounds byassays including: Caco-2 Permeability (Example 903), HepatocyteClearance (Example 904), Cytochrome P450 Inhibition (Example 905),Cytochrome P450 Induction (Example 906), Plasma Protein Binding (Example907), and hERG channel blockage (Example 908).

Certain exemplary compounds were tested for efficacy in dose escalationstudies in tumor-bearing Taconic NCR nude mouse models (Example 909).The U-87 MG Merchant (an in-house variant derived from U-87 MG cellsfrom ATCC, Manassas, Va.) subcutaneous xenograft mouse model wasemployed to test Formula I compounds at escalating doses along withVehicle (MCT, negative control). Tumor growth delay was measuredfollowing once daily oral dosing for <28 days. Body weight change overthe course of treatment was measured as an indicator of safety. Thedose- and time-dependent pharmacokinetic and pharmacodynamic response ofdrug administration in this same subcutaneous tumor xenograft model wasalso examined (Example 913).

Blood-brain barrier penetrant [properties] potential was assessed invitro using MDCK cells stably transfected with P-glycoprotein (MDR1) orbcrp1 (Example 911). Brain penetration was determined in vivo bymeasuring compound concentrations (Example 912) and/or by measuring themodulation of the PI3K pathway (Example 913) in the brain of micefollowing a single IV or oral dose. Brain tumor efficacy was measured inExample 914 by GS-2 (human glioblastoma muliforme (GBM) engineered toexpress luciferase). The effect of once daily oral dosing on the growthof GS-2 intracranial implants was evaluated by magnetic resonanceimaging (MRI). Mice with tumor xenografts of U-87 MG cells were dosedwith drug or vehicle and samples were analyzed for PK, PD, and/or IHCanalysis (Example 915).

Exemplary Formula I compounds No. 101-118 in Table 1 were made,characterized, and tested for inhibition of PI3K alpha (IC₅₀ or K_(i)binding to p110 alpha less than 1 micromolar, μM) and selectivityaccording to the methods of this invention, and have the followingstructures and corresponding names (ChemBioDraw Ultra, Version 11.0,CambridgeSoft Corp., Cambridge Mass.).

TABLE 1 No. Structure IUPAC_Name MW 101

5-(4-morpholino-6,7-dihydro- [1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine 316.315 102

5-[(6R)-6-methyl-4-morpholino- 6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl]pyrimidin-2- amine 330.342 103

5-(7-methyl-4-morpholino-6,7- dihydro-[1,4]dioxino(2,3-d]pyrimidin-2-yl)pyrimidin-2- amine 330.342 104

5-(7,7-dimethyl-4-morpholino- 6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2- amine 344.368 105

5-[4-morpholino-7- (trifluoromethyl)-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2- yl]pyrimidin-2-amine 384.313 106

5-(4-morpholinospiro[6H- [1,4]dioxino[2,3-d]pyrimidine-7,1′-cyclopropane]-2- yl)pyrimidin-2-amine 342.353 107

5-(6-cyclopropyl-4-morpholino- 6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2- amine 356.379 108

5-(7-tert-butyl-4-morpholino- 6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2- amine 372.422 109

3-(7,7-dimethyl-4-morpholino- 6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)phenol 343.377 110

2-(2-methoxypyrimidin-5-yl)- 7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidine 359.38 111

5-(7,7-dimethyl-4-morpholino- 6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyridin-2- amine 343.38 112

N-[4-(7,7-dimethyl-4- morpholino-6H- [1,4]dioxino[2,3-d]pyrimidin-2-yl)phenyl]acetamide 384.429 113

5-(7,7-dimethyl-4-morpholino- 6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)-N-methyl- pyridin-2-amine 357.407 114

N-[3-(7,7-dimethyl-4- morpholino-6H- [1,4]dioxino[2,3-d]pyrimidin-2-yl)phenyl]methanesulfonamide 420.483 115

2-(1H-indol-5-yl)-7,7-dimethyl- 4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidine 366.414 116

5-[7-(fluoromethyl)-4- morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2- yl]pyrimidin-2-amine 348.332 117

[2-(2-aminopyrimidin-5-yl)-4- morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-7- yl]methanol 346.341 118

5-(7-cyclopropyl-4-morpholino- 6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2- amine 356.38 119

5-(8-methyl-4-morpholino-7,8- dihydro-6H-pyrimido[5,4-b][1,4]oxazin-2-yl)pyrimidin-2- amine 329.36 120

2-(2-aminopyrimidin-5-yl)-8- methyl-4-morpholino-6H-pyrimido[5,4-b][1,4]oxazin- 7(8H)-one 343.34Administration of Formula I Compounds

The Formula I compounds of the invention may be administered by anyroute appropriate to the condition to be treated. Suitable routesinclude oral, parenteral (including subcutaneous, intramuscular,intravenous, intraarterial, intradermal, intrathecal and epidural),transdermal, rectal, nasal, topical (including buccal and sublingual),vaginal, intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with e.g. the conditionof the recipient. Where the compound is administered orally, it may beformulated as a pill, capsule, tablet, etc. with a pharmaceuticallyacceptable carrier or excipient. Where the compound is administeredparenterally, it may be formulated with a pharmaceutically acceptableparenteral vehicle and in a unit dosage injectable form, as detailedbelow.

A dose to treat human patients may range from about 10 mg to about 1000mg of Formula I compound. A typical dose may be about 100 mg to about300 mg of the compound. A dose may be administered once a day (QID),twice per day (BID), or more frequently, depending on thepharmacokinetic and pharmacodynamic properties, including absorption,distribution, metabolism, and excretion of the particular compound. Inaddition, toxicity factors may influence the dosage and administrationregimen. When administered orally, the pill, capsule, or tablet may beingested daily or less frequently for a specified period of time. Theregimen may be repeated for a number of cycles of therapy.

Methods of Treatment with Formula I Compounds

Formula I compounds of the present invention are useful for treatinghyperproliferative diseases, conditions and/or disorders including, butnot limited to, those characterized by over expression of lipid kinases,e.g. PI3 kinase. Accordingly, an aspect of this invention includesmethods of treating or preventing diseases or conditions that can betreated or prevented by inhibiting lipid kinases, including PI3. In oneembodiment, the method comprises administering to a mammal in needthereof a therapeutically effective amount of a compound of Formula I,or a stereoisomer, geometric isomer, tautomer, or pharmaceuticallyacceptable salt thereof. In one embodiment, a human patient is treatedwith a compound of Formula I and a pharmaceutically acceptable carrier,adjuvant, or vehicle, wherein said compound of Formula I is present inan amount to detectably inhibit PI3 kinase activity.

One embodiment of the invention includes a method of treating cancer ina patient comprised of administering to said patient a therapeuticallyeffective amount of a compound of this invention wherein the cancer isbreast, ovary, cervix, prostate, testis, genitourinary tract, esophagus,larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma,lung, epidermoid carcinoma, large cell carcinoma, non-small cell lungcarcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone,colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, renal, pancreatic, myeloid disorders, lymphoma, hairy cells,buccal cavity, naso-pharyngeal, pharynx, lip, tongue, mouth, smallintestine, colon-rectum, large intestine, rectum, brain and centralnervous system, Hodgkin's or leukemia. In one embodiment, the cancer isa brain cancer.

In one embodiment of the invention, the method further comprisesadministering to the patient an additional therapeutic agent selectedfrom a chemotherapeutic agent, an anti-angiogenesis therapeutic agent,an anti-inflammatory agent, an immunomodulatory agent, a neurotropicfactor, an agent for treating cardiovascular disease, an agent fortreating liver disease, an anti-viral agent, an agent for treating blooddisorders, an agent for treating diabetes, and an agent for treatingimmunodeficiency disorders. In one embodiment of the invention theadditional therapeutic agent is bevacizumab.

Formula I compounds may also be useful for treating hyperproliferativediseases characterized by over expression of protein kinases such asthose encoded by PIM; the genes Pim-1, Pim-2, and Pim-3 (ProviralInsertion, Moloney) which are implicated in lymphoma and solid-tumordevelopment (Cuypers et al. (1984) Cell, vol. 37 (1) pp. 141-50; Seltenet al. (1985) EMBO J. vol. 4 (7) pp. 1793-8; van der Lugt et al. (1995)EMBO J. vol. 14 (11) pp. 2536-44; Mikkers et al. (2002) Nature Genetics,vol. 32 (1) pp. 153-9; van Lohuizen et al. (1991) Cell, vol. 65 (5) pp.737-52.

Cancers which can be treated according to the methods of this inventioninclude, but are not limited to, breast, ovary, cervix, prostate,testis, genitourinary tract, esophagus, larynx, glioblastoma,neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoidcarcinoma, large cell carcinoma, non-small cell lung carcinoma (NSCLC),small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma,pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, myeloid disorders, lymphoid disorders, hairy cells, buccalcavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine,colon-rectum, large intestine, rectum, brain and central nervous system,Hodgkin's and leukemia.

Formula I compounds may be useful for in vitro, in situ, and in vivodiagnosis or treatment of mammalian cells, organisms, or associatedpathological conditions, such as systemic and local inflammation,immune-inflammatory diseases such as rheumatoid arthritis, immunesuppression, organ transplant rejection, allergies, ulcerative colitis,Crohn's disease, dermatitis, asthma, systemic lupus erythematosus,Sjögren's Syndrome, multiple sclerosis, scleroderma/systemic sclerosis,idiopathic thrombocytopenic purpura (ITP), anti-neutrophil cytoplasmicantibodies (ANCA) vasculitis, chronic obstructive pulmonary disease(COPD), psoriasis, and for general joint protective effects.

Formula I compounds may be useful for treating conditions of the brainand central nervous system which require transport across theblood-brain barrier. Certain Formula I compounds have favorablepenetrant properties across the blood-brain barrier for delivery to thebrain. Disorders of the brain which may be effectively treated withFormula I compounds include metastatic and primary brain tumors, such asglioblastoma and melanoma.

Formula I compounds may be useful for treating ocular disorders such aswet and dry Age-related Macular Degeneration (AMD) and retina edema, bylocalized delivery to the eye. Certain Formula I compounds havefavorable properties for delivery to, and uptake into, the eye. CertainFormula I compounds may enhance efficacy and extend duration of responsefor treatment of wet AMD in combination with ranibizumab (LUCENTIS®,Genentech, Inc.) and bevacizumab (AVASTIN®, Genentech, Inc.).

Another aspect of this invention provides a compound of this inventionfor use in the treatment of the diseases or conditions described hereinin a mammal, e.g., a human, suffering from such disease or condition.Also provided is the use of a compound of this invention in thepreparation of a medicament for the treatment of the diseases andconditions described herein in a warm-blooded animal, such as a mammal,e.g. a human, suffering from such disorder.

Pharmaceutical Formulations

In order to use a Formula I compound for the therapeutic treatment(including prophylactic treatment) of mammals including humans, it isnormally formulated in accordance with standard pharmaceutical practiceas a pharmaceutical composition. According to this aspect of theinvention there is provided a pharmaceutical composition comprising acompound of this invention in association with a pharmaceuticallyacceptable diluent or carrier.

One embodiment of the invention comprises a pharmaceutical compositioncomprised of a compound of this invention and a pharmaceuticallyacceptable carrier, glidant, diluent, or excipient.

One embodiment of the invention comprises a process for making apharmaceutical composition which comprises combining a compound of thisinvention with a pharmaceutically acceptable carrier.

One embodiment of the invention includes a pharmaceutical composition asdescribed above further comprising an additional therapeutic agentselected from a chemotherapeutic agent, an anti-inflammatory agent, animmunomodulatory agent, a neurotropic factor, an agent for treatingcardiovascular disease, an agent for treating liver disease, ananti-viral agent, an agent for treating blood disorders, an agent fortreating diabetes, and an agent for treating immunodeficiency disorders.

A typical formulation is prepared by mixing a Formula I compound and acarrier, diluent or excipient. Suitable carriers, diluents andexcipients are well known to those skilled in the art and includematerials such as carbohydrates, waxes, water soluble and/or swellablepolymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents,water and the like. The particular carrier, diluent or excipient usedwill depend upon the means and purpose for which the compound of thepresent invention is being applied. Solvents are generally selectedbased on solvents recognized by persons skilled in the art as safe(GRAS) to be administered to a mammal. In general, safe solvents arenon-toxic aqueous solvents such as water and other non-toxic solventsthat are soluble or miscible in water. Suitable aqueous solvents includewater, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400,PEG 300), etc. and mixtures thereof. The formulations may also includeone or more buffers, stabilizing agents, surfactants, wetting agents,lubricating agents, emulsifiers, suspending agents, preservatives,antioxidants, opaquing agents, glidants, processing aids, colorants,sweeteners, perfuming agents, flavoring agents and other known additivesto provide an elegant presentation of the drug (i.e., a compound of thepresent invention or pharmaceutical composition thereof) or aid in themanufacturing of the pharmaceutical product (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the Formula I compound(e.g., complex with a cyclodextrin derivative or other knowncomplexation agent) is dissolved in a suitable solvent in the presenceof one or more of the excipients described above. The compound of thepresent invention is typically formulated into pharmaceutical dosageforms to provide an easily controllable dosage of the drug and to enablepatient compliance with the prescribed regimen.

The pharmaceutical composition (or formulation) may be packaged in avariety of ways depending upon the method of administering the drug.Generally, an article for distribution includes a container havingdeposited therein the pharmaceutical formulation in an appropriate form.Suitable containers are well known to those skilled in the art andinclude materials such as bottles (plastic and glass), sachets,ampoules, plastic bags, metal cylinders, and the like. The container mayalso include a tamper-proof assemblage to prevent indiscreet access tothe contents of the package. In addition, the container has depositedthereon a label that describes the contents of the container. The labelmay also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1980)16^(th) edition, Osol, A. Ed.), in the form of a lyophilizedformulation, milled powder, or an aqueous solution. Formulation may beconducted by mixing at ambient temperature at the appropriate pH, and atthe desired degree of purity, with physiologically acceptable carriers,i.e., carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8. Formulation in an acetate buffer at pH 5 is asuitable embodiment.

The compound of this invention for use herein is preferably sterile. Inparticular, formulations to be used for in vivo administration must besterile. Such sterilization is readily accomplished by filtrationthrough sterile filtration membranes.

The compound ordinarily can be stored as a solid composition, alyophilized formulation or as an aqueous solution.

The pharmaceutical compositions of the invention comprising a Formula Icompound will be formulated, dosed and administered in a fashion, i.e.,amounts, concentrations, schedules, course, vehicles and route ofadministration, consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the compound to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat the coagulation factormediated disorder. Such amount is preferably below the amount that istoxic to the host or renders the host significantly more susceptible tobleeding.

As a general proposition, the initial pharmaceutically effective amountof the Formula I compound administered parenterally per dose will be inthe range of about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg ofpatient body weight per day, with the typical initial range of compoundused being 0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, e.g., by coacervation techniques or by interfacialpolymerization, e.g., hydroxymethylcellulose or gelatin-microcapsulesand poly-(methylmethacylate) microcapsules, respectively, in colloidaldrug delivery systems (e.g., liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16^(th) edition, Osol, A. Ed. (1980).

Sustained-release preparations of Formula I compounds may be prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (e.g.,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a compound of Formula I suitable for oral administrationmay be prepared as discrete units such as pills, capsules, cachets ortablets each containing a predetermined amount of a compound of FormulaI.

Compressed tablets may be prepared by compressing in a suitable machinethe active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.

Tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, e.g., gelatincapsules, syrups or elixirs may be prepared for oral use. Formulationsof compounds of Formula I intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, e.g.,inert diluents, such as calcium or sodium carbonate, lactose, calcium orsodium phosphate; granulating and disintegrating agents, such as maizestarch, or alginic acid; binding agents, such as starch, gelatin oracacia; and lubricating agents, such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated by known techniquesincluding microencapsulation to delay disintegration and adsorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations may be applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, e.g., 0.075 to 20%w/w. When formulated in an ointment, the active ingredients may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream withan oil-in-water cream base.

If desired, the aqueous phase of the cream base may include a polyhydricalcohol, i.e., an alcohol having two or more hydroxyl groups such aspropylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol andpolyethylene glycol (including PEG 400) and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active ingredient through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethyl sulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier, it desirably comprises a mixture of at least oneemulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with an oil and fat make up theso-called emulsifying ointment base which forms the oily dispersed phaseof the cream formulations. Emulsifiers and emulsion stabilizers suitablefor use in the formulation of the invention include Tween® 60, Span® 80,cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glycerylmono-stearate and sodium lauryl sulfate.

Aqueous suspensions of Formula I compounds contain the active materialsin admixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth and gum acacia, and dispersing or wetting agents such asa naturally occurring phosphatide (e.g., lecithin), a condensationproduct of an alkylene oxide with a fatty acid (e.g., polyoxyethylenestearate), a condensation product of ethylene oxide with a long chainaliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such as sucroseor saccharin.

The pharmaceutical compositions of compounds of Formula I may be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butanediol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, about 0.5 to10% w/w, or about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising e.g. cocoa butter or a salicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size e.g. in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,e.g. sealed ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, e.g. water, for injection immediately prior to use.Extemporaneous injection solutions and suspensions are prepared fromsterile powders, granules and tablets of the kind previously described.Preferred unit dosage formulations are those containing a daily dose orunit daily sub-dose, as herein above recited, or an appropriate fractionthereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compounds of Formula I may be employed alone or in combination withother therapeutic agents for the treatment of a disease or disorderdescribed herein, such as a hyperproliferative disorder (e.g., cancer).In certain embodiments, a compound of Formula I is combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound that has anti-hyperproliferativeproperties or that is useful for treating a hyperproliferative disorder(e.g., cancer). The second compound of the pharmaceutical combinationformulation or dosing regimen preferably has complementary activities tothe compound of Formula I such that they do not adversely affect eachother. Such compounds are suitably present in combination in amountsthat are effective for the purpose intended. In one embodiment, acomposition of this invention comprises a compound of Formula I, incombination with a chemotherapeutic agent such as described herein.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes,separate pills or capsules, or separate infusions. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

In a particular embodiment of anti-cancer therapy, a compound of FormulaI, or a stereoisomer, geometric isomer, tautomer, solvate, metabolite,or pharmaceutically acceptable salt or prodrug thereof, may be combinedwith other chemotherapeutic, hormonal or antibody agents such as thosedescribed herein, as well as combined with surgical therapy andradiotherapy. Combination therapies according to the present inventionthus comprise the administration of at least one compound of Formula I,or a stereoisomer, geometric isomer, tautomer, solvate, metabolite, orpharmaceutically acceptable salt or prodrug thereof, and the use of atleast one other cancer treatment method. The amounts of the compound(s)of Formula I and the other pharmaceutically active chemotherapeuticagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect.

Metabolites of Formula I Compounds

Also falling within the scope of this invention are the in vivometabolic products of Formula I described herein. Such products mayresult e.g. from the oxidation, reduction, hydrolysis, amidation,deamidation, esterification, deesterification, enzymatic cleavage, andthe like, of the administered compound. Accordingly, the inventionincludes metabolites of compounds of Formula I, including compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, may be useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the diseases anddisorders described above is provided. The kit comprises a containercomprising a compound of Formula I. The kit may further comprise a labelor package insert, on or associated with the container. The term“package insert” is used to refer to instructions customarily includedin commercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.

One embodiment of the invention comprises a kit for treating aPI3K-mediated condition, comprising a compound of this invention, andinstructions for use.

Suitable containers include, e.g., bottles, vials, syringes, blisterpack, etc. The container may be formed from a variety of materials suchas glass or plastic. The container may hold a compound of Formula I or aformulation thereof which is effective for treating the condition andmay have a sterile access port (e.g., the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is a compound of Formula I. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. In addition, the label or package insert mayindicate that the patient to be treated is one having a disorder such asa hyperproliferative disorder, neurodegeneration, cardiac hypertrophy,pain, migraine or a neurotraumatic disease or event. In one embodiment,the label or package inserts indicates that the composition comprising acompound of Formula I can be used to treat a disorder resulting fromabnormal cell growth. The label or package insert may also indicate thatthe composition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I, and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, e.g. in the form of numbers, letters, or other markings orwith a calendar insert, designating the days in the treatment schedulein which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I contained therein; and optionally (b) asecond container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain other embodiments wherein the kit comprises a composition ofFormula I and a second therapeutic agent, the kit may comprise acontainer for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

Preparation of Formula I Compounds

Tricyclic compounds of Formula I may be synthesized by synthetic routesthat include processes analogous to those well-known in the chemicalarts, particularly in light of the description contained herein. Thestarting materials are generally available from commercial sources suchas Aldrich Chemicals (Milwaukee, Wis.) or are readily prepared usingmethods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-23, Wiley, N.Y. (1967-2006 ed.), orBeilsteins Handbuch der organischen Chemie, 4, Aufl. ed.Springer-Verlag, Berlin, including supplements (also available via theBeilstein online database).

In certain embodiments, compounds of Formula I may be readily preparedusing well-known procedures to prepare heterocycle compounds describedin: Comprehensive Heterocyclic Chemistry II, Editors Katritzky and Rees,Elsevier, 1997, e.g. Volume 3; Liebigs Annalen der Chemie, (9):1910-16,(1985); Helvetica Chimica Acta, 41:1052-60, (1958);Arzneimittel-Forschung, 40(12):1328-31, (1990).

Compounds of Formula I may be prepared singly or as compound librariescomprising at least 2, e.g. 5 to 1,000 compounds, or 10 to 100compounds. Libraries of compounds of Formula I may be prepared by acombinatorial ‘split and mix’ approach or by multiple parallel synthesesusing either solution phase or solid phase chemistry, by proceduresknown to those skilled in the art. Thus according to a further aspect ofthe invention there is provided a compound library comprising at least 2compounds, or pharmaceutically acceptable salts thereof.

For illustrative purposes, the General Procedures show general methodswhich may be applied for preparation of Formula I compounds, as well askey intermediates. The Figures and Examples sections contain moredetailed description of individual reaction steps. Those skilled in theart will appreciate that other synthetic routes may be used tosynthesize the inventive compounds. Although certain starting materialsand routes are depicted in the Schemes, General Procedures and Examples,other similar starting materials and routes can be substituted toprovide a variety of derivatives and/or reaction conditions. Inaddition, many of the compounds prepared by the methods described belowcan be further modified in light of this disclosure using conventionalchemistry well known to those skilled in the art.

In preparing compounds of Formula I, protection of remote functionality(e.g., primary or secondary amine) of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, Third Ed., 1999.

Methods of Separation

In the methods of preparing the compounds of this invention, it may beadvantageous to separate reaction products from one another and/or fromstarting materials. The desired products of each step or series of stepsis separated and/or purified (hereinafter separated) to the desireddegree of homogeneity by the techniques common in the art. Typicallysuch separations involve multiphase extraction, crystallization from asolvent or solvent mixture, distillation, sublimation, orchromatography. Chromatography can involve any number of methodsincluding, e.g.: reverse-phase and normal phase; size exclusion; ionexchange; high, medium and low pressure liquid chromatography methodsand apparatus; small scale analytical; simulated moving bed (SMB) andpreparative thin or thick layer chromatography, as well as techniques ofsmall scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. For example, boiling point and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-S phenylethylamine (amphetamine), and the likewith asymmetric compounds bearing acidic functionality, such ascarboxylic acid and sulfonic acid. The diastereomeric salts may beinduced to separate by fractional crystallization or ionicchromatography. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters of the racemic mixture, such as a menthyl ester,for example with (−) menthyl chloroformate, in the presence of base, orMosher ester, α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J.Org. Chem. (1982) 47:4165), and analyzing the ¹H NMR spectrum for thepresence of the two atropisomeric enantiomers or diastereomers. Stablediastereomers of atropisomeric compounds can be separated and isolatedby normal- and reverse-phase chromatography following methods forseparation of atropisomeric naphthyl-isoquinolines (WO 1996/015111). Bymethod (3), a racemic mixture of two enantiomers can be separated bychromatography using a chiral stationary phase (“Chiral LiquidChromatography” (1989) W. J. Lough, Ed., Chapman and Hall, New York;Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

General Preparative Procedures

Suzuki Coupling:

The Suzuki-type coupling reaction is useful to attach a monocyclicheteroaryl, a fused bicyclic heterocycle, a fused bicyclic heteroaryl,or a phenyl at the 2-position of the pyrimidine ring of a2-chloro-pyrimidine 1 intermediate of Formula I compounds. For example,1 may be combined with an aryl or heteroaryl boronate reagent 2 where Ris H or a protected boronate such as pinacol. For example, 1.5equivalents of4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1H-indazole 24 isdissolved in about 3 equivalents of sodium carbonate as about a 1 molarsolution in water and about an equal volume of acetonitrile. A catalyticamount, or more, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II)dichloride, is added to give 3.Substituents R^(1′), R^(2′), R^(3′) may be R¹, R², R³ as defined, orprotected forms or precursors thereof.

A variety of boronic acids or boronic esters can be used in place of theindazole boronic ester indicated. Also alternatively, the nitrogen ofthe indazole may be protected, for example, N-THP protected compound 25.In some cases potassium acetate is used in place of sodium carbonate toadjust the pH of the aqueous layer. The Suzuki palladium couplingreaction may be optimized and/or accelerated under microwave conditions.The reaction may be heated at about 100-150° C. under pressure in amicrowave reactor such as the Biotage Optimizer (Biotage, Inc.) forabout 10 to 30 minutes. The contents are cooled, concentrated, andextracted with ethyl acetate, or another organic solvent. Afterevaporation of the organic layer the Suzuki coupling products 3, may bepurified on silica or by reverse phase HPLC.

A variety of palladium catalysts can be used during palladium mediatedcross Suzuki coupling reaction of a halide 1 with a boronic acid orester 2, such as4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole 24 or4-methyl-5-(4,4,5,5-tetramethyl(1,3,2-dioxaborolan-2-yl))pyrimidine-2-ylamine26. Low valent, Pd(II) and Pd(0) catalysts may be used in the Suzukicoupling reaction, including PdCl2(PPh₃)₂, Pd(t-Bu)₃, PdCl₂ dppf CH₂Cl₂,Pd(PPh₃)₄, Pd(OAc)/PPh₃, Cl₂Pd[(Pet₃)]₂, Pd(DIPHOS)₂, Cl₂Pd(Bipy),[PdCl(Ph₂PCH₂PPh₂)]₂, Cl₂Pd[P(o-tol)₃]₂, Pd₂(dba)₃/P(o-tol)₃,Pd₂(dba)/P(furyl)₃, Cl₂Pd[P(furyl)₃]₂, Cl₂Pd(PmePh₂)₂,Cl₂Pd[P(4-F-Ph)₃]₂, Cl₂Pd[P(C₆F₆)₃]₂, Cl₂Pd[P(2-COOH- Ph)(Ph)₂]₂,Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂, and encapsulated catalysts Pd EnCat™ 30, PdEnCat™ TPP30, and Pd(II)EnCat™ BINAP30 (US 2004/0254066). One suchSuzuki palladium catalyst is[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane, represented as Pd (dppf)Cl₂.

EXAMPLES

The chemical reactions described in the Examples may be readily adaptedto prepare a number of other PI3K inhibitors of the invention, andalternative methods for preparing the compounds of this invention aredeemed to be within the scope of this invention. For example, thesynthesis of non-exemplified compounds according to the invention may besuccessfully performed by modifications apparent to those skilled in theart, e.g., by appropriately protecting reactive functional groups, byutilizing other suitable reagents known in the art other than thosedescribed, and/or by making routine modifications of reactionconditions. Alternatively, other reactions disclosed herein or known inthe art will be recognized as having applicability for preparing othercompounds of the invention.

In the Examples described below, unless otherwise indicated alltemperatures are set forth in degrees Celsius. Reagents were purchasedfrom commercial suppliers such as Sigma Aldrich Chemical Company,Lancaster, TCI or Maybridge, and were used without further purificationunless otherwise indicated. The reactions set forth below were donegenerally under a positive pressure of nitrogen or argon or with adrying tube (unless otherwise stated) in anhydrous solvents, and thereaction flasks were typically fitted with rubber septa for theintroduction of substrates and reagents via syringe. Glassware was ovendried and/or heat dried. Column chromatography was conducted on aBiotage system (Manufacturer: Dyax Corporation) having a silica gelcolumn or on a silica SEP PAK® cartridge (Waters). ¹H NMR spectra wereobtained at 400 MHz in deuterated CDCl₃, d₆-DMSO, CH₃OD or d₆-acetonesolutions (reported in ppm), using chloroform as the reference standard(7.25 ppm). When peak multiplicities are reported, the followingabbreviations are used: s (singlet), d (doublet), t (triplet), m(multiplet), br (broadened), dd (doublet of doublets), dt (doublet oftriplets). Coupling constants, when given, are reported in hertz (Hz).

HPLC was conducted by the following exemplary methods:

(A) LCMS short method - 10 min run HPLC-Agilent 1200 Mobile phase AWater with 0.05% TFA Mobile phase B Acetonitrile with 0.05% TFA AgilentZORBAX SD-C18, 1.8 μm, Column 2.1 × 30 mm Column temperature 40 degreeC. LC gradient 3-95% B in 8.5 min, 95% in 2.5 min LC Flowrate 400 μL/minUV wavelength 220 nm and 254 nm Mass Spec - Agilent quadrupole 6140Ionization ESI positive Scan range 110-800 amu

(B) Waters Acquity/LCT long method - 20 min run Waters Acquity UPLCMobile phase A Waters with 0.05% TFA Mobile phase B Acetonitrile with0.05% TFA Acquity UPLC BEH C18, 1.7 μm, Column 2.1 × 50 mm Columntemperature 40 degree C. LC gradient 2-98% B in 17.0 min, 98% in 1.5 minLC Flowrate 600 μL/min UV wavelength 254 nm Mass Spec - Waters LCTPremier XE Ionization ESI positive Scan range 100-800 amu(C) LCMS 2.5 min Chiral methodMobile Phase A: CO2Mobile Phase B: methanolIsocratic conditions: 25% BFlow rate: 5 mL/minOutlet pressure: 120 BarTemperature: 40° C.Column: ChiralCel OJ (4.6×50 mm, 3 μm)Uv: 230 nmSystem: Berger Analytical SFC/MSChiral purification:Conditions A:Mobile Phase A: CO2Mobile Phase B: methanolIsocratic conditions: 25% BFlow rate: 60 mL/minOutlet pressure: 100 BarTemperature: 40 degrees C.Column: ChiralCel OJ (21.2×250 mm, 5 μm)Uv: 230 nmSystem: Berger MGII

Example 1 6-Hydroxy-5-methoxy-1H-pyrimidine-2,4-dione, sodium salt

Under an atmosphere of nitrogen, sodium metal (1.15 g, 0.05 moles) wasadded portionwise to ethanol (dried over 4 Å molecular sieves, 50 mL) at40° C. and the mixture stirred until a solution was formed. Urea (3.0 g,0.05 moles) was added and the mixture heated at 100° C. for 15 minutesuntil complete dissolution was achieved. The reaction mixture wasallowed to cool slightly and methoxymethyl malonate (8.1 g, 0.05 moles)added, a pink/white precipitate forming almost immediately. Further dryethanol (10 mL) was added to maintain a stirrable mixture. The resultantsuspension was heated at 100° C. (reflux) for 4 hours. The reactionmixture was concentrated in vacuo and the residue dried under highvacuum to give 6-Hydroxy-5-methoxy-1H-pyrimidine-2,4-dione, sodium saltas a pink/white solid, used in the subsequent step without analysis orpurification.

Example 2 2,4,6-Trichloro-5-methoxy-pyrimidine

6-Hydroxy-5-methoxy-1H-pyrimidine-2,4-dione, sodium salt (21 mmol) wassuspended in phosphorous oxychloride (20 mL) and the mixture dividedbetween two 20 mL microwave reaction vials. The reaction mixtures wereheated at 130-140° C. (−10-12 bar) for 30 minutes using microwaveirradiation (CARE! SIGNIFICANT PRESSURE INCREASE!). The cooled reactionmixtures were combined and poured onto warm (approximately 40° C.) water(CARE!) and the resultant mixture extracted twice with ethyl acetate,the combined organic extracts were dried (Na₂SO₄), filtered andconcentrated in vacuo to give 2,4,6-Trichloro-5-methoxy-pyrimidine as acrystalline yellow/brown solid (3.75 g, 84%). ¹H NMR (CDCl₃): 3.98 (3H,s).

Example 3 2,4,6-Trichloro-5-hydroxy-pyrimidine

Under an atmosphere of nitrogen a solution of2,4,6-trichloro-5-methoxy-pyrimidine (4.0 g, 18.7 mmol) in DCM (200 mL)was cooled to 0° C. and treated with boron tribromide (6.6 mL, 65 mmol)dropwise. After stirring for 18 hours at RT the reaction mixture wascooled and diluted with methanol (25 mL, CARE, EXOTHERM!) and thereaction mixture diluted with water (200 mL). The aqueous layer wasextracted with DCM and the combined organic extracts dried (Na₂SO₄) andconcentrated in vacuo to give 2,4,6-Trichloro-5-hydroxy-pyrimidine as apale tan solid (2.55 g, 71%). ¹³C NMR (DMSO-d₆): 149.23 (C), 145.25 (C),145.08 (C). LCMS (Method C): R_(T)=2.65/2.77. [M−H]⁺ 197/199.

Example 4 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole24-route 1

To a solution of 3-bromo-2-methyl aniline (5.0 g, 26.9 mmol) inchloroform (50 mL) was added potassium acetate (1.05 eq., 28.2 mmol,2.77 g). Acetic anhydride (2.0 eq., 53.7 mmol, 5.07 mL) was added withconcurrent cooling in ice-water. The mixture was then stirred at roomtemperature for 10 minutes after which time a white gelatinous solidformed. 18-Crown-6 (0.2 eq., 5.37 mmol, 1.42 g) was added followed byiso-amyl nitrite (2.2 eq., 59.1 mmol, 7.94 mL) and the mixture washeated under reflux for 18 h. The reaction mixture was allowed to cool,and was partitioned between chloroform (3×100 mL) and saturated aqueoussodium hydrogen carbonate (100 mL). The combined organic extracts werewashed with brine (100 mL), separated and dried (MgSO₄). The crudeproduct was evaporated onto silica and purified by chromatographyeluting with 20% to 40% EtOAc-petrol to give1-(4-bromo-indazol-1-yl)-ethanone A (3.14 g, 49%) as an orange solid,and 4-bromo-1H-indazole B (2.13 g, 40%) as a pale orange solid. A: ¹HNMR (400 MHz, CDCl₃) δ 2.80 (3H, s), 7.41 (1H, t, J=7.8 Hz), 7.50 (1H,d, J=7.8 Hz), 8.15 (1H, s), 8.40 (1H, d, J=7.8 Hz). B: ¹H NMR (400 MHz,CDCl₃) 7.25 (1H, t, J=7.3 Hz), 7.33 (1H, d, J=7.3 Hz), 7.46 (1H, d,J=7.3 Hz), 8.11 (1H, s), 10.20 (1H, br s).

To a solution of the 1-(4-bromo-indazol-1-yl)-ethanone A (3.09 g, 12.9mmol) in MeOH (50 mL) was added 6N aqueous HCl (30 mL) and the mixturewas stirred at room temperature for 7 h. The MeOH was evaporated and themixture partitioned between EtOAc (2×50 mL) and water (50 mL). Thecombined organic layers were washed with brine (50 mL), separated anddried (MgSO₄). The solvent was removed by evaporation under reducedpressure to give 4-bromo-1H-indazole B (2.36 g, 93%).

To a solution of the 4-bromo-1H-indazole B (500 mg, 2.54 mmol) andbis(pinacolato)diboron (1.5 eq., 3.81 mmol) in DMSO (20 mL) was addedpotassium acetate (3.0 eq., 7.61 mmol, 747 mg; dried in drying pistol)and PdCl₂(dppf)₂ (3 mol %, 0.076 mmol, 62 mg). The mixture was degassedwith argon and heated at 80° C. for 40 h. The reaction mixture wasallowed to cool and partitioned between water (50 mL) and ether (3×50mL). The combined organic layers were washed with brine (50 mL),separated and dried (MgSO₄). The crude material was purified bychromatography eluting with 30% to 40% EtOAc-petrol to give aninseparable 3:1 mixture of the4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole 24 (369 mg,60%) and indazole (60 mg, 20%), isolated as a yellow gum whichsolidified upon standing to furnish as an off-white solid. ¹H NMR (400MHz, d₆-DMSO) 1.41 (12H, s), 7.40 (1H, dd, J=8.4 Hz, 6.9 Hz), 7.59 (1H,d, J=8.4 Hz), 7.67 (1H, d, J=6.9 Hz), 10.00 (1H, br s), 8.45 (1H, s),and indazole: 7.40 (1H, t), 7.18 (1H, t, J=7.9 Hz), 7.50 (1H, d, J=9.1Hz), 7.77 (1H, d, J=7.9 Hz), 8.09 (1H, s); impurity at 1.25.

Example 5 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole24-route 2

To a solution of 2-methyl-3-nitroaniline (2.27 g, 14.91 mmol) in aceticacid (60 mL) was added a solution of sodium nitrite (1.13 g, 1.1 eq.) inwater (5 mL). After 2 h, the deep red solution was poured onto ice/waterand the resulting precipitate collected by filtration to yield4-nitro-1H-indazole C (1.98 g, 81%).

A mixture of 4-nitro-1H-indazole C (760 mg, 4.68 mmol), palladium oncharcoal (10%, cat.) and ethanol (30 mL) was stirred under a balloon ofhydrogen for 4 h. The reaction mixture was then filtered through celite,and the solvent removed in vacuo to yield 1H-indazol-4-ylamine D (631mg, 100%).

An aqueous solution of sodium nitrite (337 mg, 4.89 mmol) in water (2mL) was added dropwise to a suspension of 1H-indazol-4-ylamine D (631mg, 4.74 mmol) in 6M hydrochloric acid (7.2 mL) at below 0° C. Afterstirring for 30 minutes, sodium tetrafluoroborate (724 mg) was added tothe reaction mixture. A viscous solution resulted, which was filteredand washed briefly with water to yield 1H-indazole-4-diazoniumtetrafluoroborate salt E (218 mg, 20%) as a deep red solid.

Dry methanol (4 mL) was purged with argon for 5 minutes. To this wasadded 1H-indazole-4-diazonium tetrafluoroborate salt (218 mg, 0.94mmol), bis-pinacolato diboron (239 mg, 1.0 eq.) and[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride (20 mg).The reaction mixture was stirred for 5 h and then filtered throughcelite. The residue was purified using flash chromatography to yield4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole 24 (117mg).

Example 61-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole25 (Route A)

Step A: Preparation of 4-chloro-1H-indazole: To a 250 ml flask with stirbar was added 2-methyl-3-chloroaniline (8.4 ml, 9.95 g, 70.6 mmol),potassium acetate (8.3 g, 84.7 mmol) and chloroform (120 ml). Thismixture was cooled to 0° C. with stirring. To the cooled mixture wasadded acetic anhydride (20.0 ml, 212 mmol) drop wise over 2 minutes. Thereaction mixture was warmed to 25° C. and stirred for 1 hour. At thispoint, the reaction was heated to 60° C. Isoamyl nitrite (18.9 ml, 141mmol) was added and the reaction was stirred overnight at 60° C. Oncecomplete, water (75 ml) and THF (150 ml) were added and the reaction wascooled to 0° C. LiOH (20.7 g, 494 mmol) was added and the reaction wasstirred at 0° C. for 3 hours. Water (200 ml) was added and the productwas extracted with EtOAc (300 ml, 100 ml). The organic layers werecombined, dried with MgSO₄ and concentrated in vacuo to yield4-chloro-1H-indazole 11.07 g (100%) as an orange solid. ¹H NMR (400 MHz,CDCl₃) δ 8.18 (d, J=1 Hz, 1H), 7.33 (d, J=8 Hz 1H), 7.31 (t, J=7 Hz,1H), 7.17 (dd, J=7 Hz, 1 Hz 1H). LCMS (ESI pos) m/e 153 (M+1).

Step B: Preparation of4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole: To a 1 L flask withmechanical stirrer was added 4-chloro-1H-indazole (75.0 g, 0.492 mol),pyridinium p-toluenesulfonate (1.24 g, 4.92 mmol), CH₂Cl₂ (500 ml) and3,4-dihydro-2H-pyran (98.6 ml, 1.08 mol). With stirring, this mixturewas heated to 45° C. for 16 hours. Analysis of reaction mixture showsproduction of both isomers of product. Cooled reaction to 25° C. andadded CH₂Cl₂ (200 ml). Washed the solution with water (300 ml) andsaturated NaHCO₃ (250 ml). Dried the organics with MgSO₄ andconcentrated to dryness. Purified the crude product by dissolving inEtOAc/hexanes (4:6, 1 L) and adding SiO₂ (1.2 L). The mixture wasfiltered and the cake was washed with EtOAc/Hexanes (4:6, 2 L). Theorganics were concentrated in vacuo to yield4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole 110.2 g (95%) as anorange solid. Isomer 1: ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J=1 Hz, 1H),7.50 (dd, J=9 Hz, 1 Hz 1H), 7.29 (dd, J=9 Hz, 8 Hz 1H), 7.15 (dd, J=8Hz, 1 Hz 1H) 5.71 (dd, J=9 Hz, 3 Hz 1H) 4.02 (m, 1H) 3.55 (m, 1H) 2.51(m, 1H) 2.02 (m, 2H) 1.55 (m, 3H). LCMS (ESI pos) m/e 237 (M+1); Isomer2: ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J=1 Hz, 1H), 7.62 (dd, J=9 Hz, 1Hz 1H), 7.20 (dd, J=9 Hz, 8 Hz 1H), 7.06 (dd, J=8 Hz, 1 Hz 1H) 5.69 (dd,J=9 Hz, 3 Hz 1H) 4.15 (m, 1H) 3.80 (m, 1H) 2.22 (m, 2H) 2.05 (m, 1H)1.75 (m, 3H). LCMS (ESI pos) m/e 237 (M+1).

Step C: Preparation of1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole:To a 500 ml flask with stir bar was added4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (10.0 g, 42.2 mmol),DMSO (176 ml), PdCl₂(PPh₃)₂ (6.2 g, 8.86 mmol), tricyclohexylphosphine(0.47 g, 1.69 mmol), bis(pinacolato)diboron (16.1 g, 63.4 mmol) andpotassium acetate (12.4 g, 0.127 mol). With stirring, the mixture washeated to 130° C. for 16 hours. The reaction was cooled to 25° C. andEtOAc (600 ml) was added and washed with water (2×250 ml). The organicswere dried with MgSO₄ and concentrated in vacuo to dryness. The crudeproduct was purified by SiO₂ plug (120 g), eluting with 10%EtOAc/Hexanes (1 L) and 30% EtOAc/Hexanes (1 L). The filtrate wasconcentrated in vacuo to give 13.9 g (100%) of1-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazoleas a 20% (wt/wt) solution in ethyl acetate. ¹H NMR shows the presence ofabout 20% (wt/wt) bis(pinacolato)diboron. ¹H NMR (400 MHz, CDCl₃) δ 8.37(s, 1H), 7.62 (dd, J=14 Hz, 2 Hz 1H), 7.60 (dd, J=7 Hz, 1 Hz 1H), 7.31(dd, J=8 Hz, 7 Hz 1H) 5.65 (dd, J=9 Hz, 3 Hz 1H) 4.05 (m, 1H) 3.75 (m,1H) 2.59 (m, 1H) 2.15 (m, 1H) 2.05 (m, 1H) 1.75 (m, 3H) 1.34 (s, 12H).LCMS (ESI pos) m/e 245 (M+1).

Example 7 1-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1H-indazole 25 (Route B)

Step A: Preparation of 4-nitro-1H-indazole: A mixture of2-methyl-3-nitro aniline (200 g, 1.315 moles), acetic acid (8000 ml) wascooled to 15-20° C. and a solution of sodium nitrite (90.6 g, 1.315moles) in water (200 ml) was slowly added over 30 min. After theaddition, the reaction temp. was increased to 25-30° C. and the reactionwas stirred at this temp for 2-3 h. Reaction progress was monitored byTLC and after completion of reaction product was filtered and residuewas washed with acetic acid (1000 ml). Acetic acid was distilled undervacuum (550 mm of Hg) below 80° C. and water (8000 ml) was added, cooledto 25-30° C. and stirred for 30 min. The slurry was filtered and washedwith water (1000 ml). Crude product was dried under heating at 70-80° C.for 2 hours, then was taken in 5% ethyl acetate/n-hexane (100:2000 ml)solution and stirred for 1-1.5 h at ambient temperature. The suspensionwas filtered and washed with 5% ethyl acetate/n-hexane mixture (25:475ml). The product obtained was dried under vacuum at below 80° C. for10-12 h to give 4-nitro-1H-indazole as a brown solid (150 g, 70%): mp:200-203° C.; ¹H NMR (200 MHz, CDCl₃) δ 13.4 (br, 1H), 8.6 (s, 1H),8.2-7.95 (dd, 2H), 7.4 (m, 1H). ESMS m/z 164 (M+1). Purity: 95% (HPLC)

Step B: Preparation of 4-amino-1H-indazole: A mixture of4-nitro-1H-indazole (200 g, 1.22 moles) and 10% palladium on carbon(20.0 g,) in EtOH (3000 ml) was hydrogenated at ambient temperature(reaction was exothermic and temperature increased to 50° C.). Aftercompletion of reaction, the catalyst was removed by filtration. Thesolvent was evaporated under vacuum at below 80° C. and cooled to roomtemperature and n-hexane (1000 ml) was added to the residue and stirredfor 30 min. Isolated solid was filtered and washed with n-hexane (200ml). Product was dried under vacuum at 70-80° C. for 10-12 h to give4-amino-1H-indazole as a brown solid (114 g, 70%), m. p.: 136-143° C. ¹HNMR (200 MHz, CDCl₃) δ 12 (br, 1H), 8.0 (s, 1H), 7.1-7.0 (dd, 2H), 6.5(d, 1H), 3.9 (m, 2H). ESMS m/z 134 (M+1). Purity: 90-95% (HPLC)

Step C: Preparation of 4-iodo-1H-indazole: A mixture of4-amino-1H-indazole (50.0 g, 0.375 moles) in water (100 ml) and con.hydrochloric acid (182 ml) was cooled to −10° C. To this a solution ofsodium nitrite (51.7 g, 0.75 moles) in water (75 ml) was added drop wiseat −10° C. in about 30-60 min. (during addition frothing was observed).In another flask a mixture of potassium iodide (311 g, 1.87 moles) inwater (3000 ml) was prepared at room temperature and to this abovecooled diazonium salt at 30-40° C. was added in about 30-40 min. Thereaction was maintained at 30° C. for 1 h and after completion ofreaction, ethyl acetate (500 ml) was added and the reaction mixture wasfiltered through Celite. The layers were separated and the aq. layer wasextracted with ethyl acetate (2×500 ml). The combined organic layerswere washed with 5% hypo solution (2×500 ml), brine (500 ml), dried(Na₂SO₄) and concentrated. Crude product was purified by chromatography(silica gel, hexane, 15-20% ethyl acetate/hexane) to furnish4-iodo-1H-indazole as an orange solid (23.0 g, 25%). mp: 151-177 C: ¹HNMR (200 MHz, CDCl₃) δ 12.4 (br, 1H), 8.0 (s, 1H), 7.6 (dd, 2H), 7.1 (d,1H). ESMS m/z 245 (M+1). Purity: 95-98% (HPLC).

Step D: Preparation of 4-iodo-1-(2-tetrahydropyranyl) indazole: Amixture of 4-amino-1H-indazole (250.0 g, 1.024 moles),3,4-dihydro-2H-pyran (126.0 g, 1.5 moles) and PPTS (2.57 g, 0.01 moles)in CH₂Cl₂ (1250 ml) was heated to 50° C. for 2 h. The reaction wascooled to room temperature and poured into water (625 ml), the layerswere separated, and aqueous layer was extracted with CH₂Cl₂ (250 ml).The combined organic layers were washed with water (625 ml), dried(Na₂SO₄) and concentrated. Crude residue was purified by chromatography(silica gel, hexane, 5-10% ethyl acetate/hexane) to furnish4-iodo-1-(2-tetrahydropyranyl) indazole as an oil (807.0 g, 60%). ¹H NMR(200 MHz, CDCl₃) δ 8.5 (s, 1H), 7.8 (m, 1H), 7.6 (d, 1H), 7.25 (m, 1H),5.7 (dd, 1H), 4.2-3.8 (dd, 1H), 2.2-2.0 (m, 4H) 2.0-1.8 (m, 4H). ESMSm/z 329 (M+1).

Step E: Preparation of1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole:A mixture of 4-iodo-1-(2-tetrahydropyranyl) indazole (100 g, 0.304moles), bispinacalotodiborane (96.4 g, 0.381 moles), PdCl₂ (dppf) (8.91g, 0.012 moles) and potassium acetate (85.97 g, 0.905 moles) in DMSO(500 ml) were heated to 80° C. for 2-3 h. After completion, reaction wascooled to room temperature and water (1500 ml) was added. Reaction masswas extracted into ethyl acetate (3×200 ml) and combined organic layerswere evaporated, dried (Na₂SO₄) and concentrated. Crude product waspurified by column chromatography (silica gel, hexane, 5-10% ethylacetate/hexane) to obtain1-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole25 as viscous brown oil (70.0 g, 70%). ¹H NMR (CDCl₃) δ 8.5 (s, 1H), 7.8(m, 1H), 7.6 (d, 1H), 7.25 (m, 1H), 5.7 (dd, 1H), 4.2-3.8 (dd, 1H),2.2-2.0 (m, 4H) 2.0-1.8 (m, 4H) 1.4-1.2 (s, 12H). ESMS m/z 329 (M+1)

Example 84-methyl-5-(4,4,5,5-tetramethyl(1,3,2-dioxaborolan-2-yl))pyrimidine-2-ylamine26

To a solution of 4-methylpyrimidine-2-ylamine (8.0 g, 0.073 mol) inchloroform (320 mL) was added N-bromosuccinimide (13.7 g, 0.077 mol).The reaction mixture was stirred in the dark for 18 hrs. LC/MS indicatedthe reaction was completed. The mixture was diluted with DCM, thenwashed with 1N NaOH aq solution and brine, dried over MgSO₄, filteredand concentrated to yield 5-bromo-4-methylpyrimidine-2-ylamin (12 g,Yield: 86%).

A mixture of 5-bromo-4-methylpyrimidine-2-ylamine (5.0 g, 26 mmol),potassium acetate (7.83 g, 79.8 mmol),4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane(7.43 g, 29.2 mmol) in dioxane (140 mL) was stirred for 20 min undernitrogen. 1,1′-bis(diphenylphosphino)ferrocene palladium (II) chloridedichloromethane adduct (1.08 g, 1.33 mmol) was added to the reactionmixture. The reaction mixture was heated to 115° C. for 18 h undernitrogen. Upon completion, the mixture was cooled and EtOAc was added.The resulting mixture was sonicated and filtered. Additional EtOAc wasused to wash the solid. The combined organic extracts were washed withwater, dried over MgSO₄, filtered and concentrated. The crude waspurified by chromatography eluting with 20˜100% EtOAc/hexane to yield4.5 g of4-methyl-5-(4,4,5,5-tetramethyl(1,3,2-dioxaborolan-2-yl))pyrimidine-2-ylamine26 (yield: 74%). ¹H-NMR (DMSO, 400 MHz): δ 8.28 (s, 1H), 6.86 (br s,2H), 2.35 (s, 3H), 1.25 (s, 12H). MS (ESI) m/e (M+H⁺) 236.15, 154.07.

Example 1015-(4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine101

Step 1: (2,4,6-Trichloro-pyrimidin-5-yloxy)-acetic acid ethyl ester

A mixture of 2,4,6-trichloro-pyrimidin-5-ol (3.0 g, 15.0 mmol),ethylglycolate (16.7 mL, 17.3 mmol), triphenylphosphine (4.5 g, 17.3mmol) and DIAD (3.4 mL, 17.3 mmol) in dioxane (100 mL) was stirred at RTfor 18 hours then concentrated in vacuo. The resulting residue waspurified by column chromatography (SiO₂, gradient 0-15% ethyl acetate incyclohexane) affording (2,4,6-Trichloro-pyrimidin-5-yloxy)-acetic acidethyl ester as a white solid (2.67 g, 62%). ¹H NMR (400 MHz, CDCl₃): δ4.77 (2H, s), 4.28 (2H, q, J=7.14 Hz), 1.32 (3H, t, J=7.15 Hz).

Step 2: (2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-acetic acidethyl ester

A mixture of (2,4,6-trichloro-pyrimidin-5-yloxy)-acetic acid ethyl ester(2.67 g, 9.35 mmol), morpholine (815 μL, 9.35 mmol) and triethylamine(2.6 mL, 18.7 mmol) in IMS (50 mL) was stirred at RT for 2 hours beforethe reaction was concentrated in vacuo. The resultant residue waspartitioned between ethyl acetate and saturated aqueous sodiumbicarbonate solution. The aqueous phase was extracted with further ethylacetate and the combined organic extracts dried (Na₂SO₄) andconcentrated in vacuo affording(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-acetic acid ethylester (2.97 g, 95%). LCMS: R_(T)=3.35 min, [M+H]⁺=336/338/340.

Step 3: 2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanol

To a solution of(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-acetic acid ethylester (188 mg, 0.56 mmol) in THF (2.5 mL) at −78° C. was added DIBAL-H(2.0 mL, 2.0 mmol, 1M solution in THF) and the resulting mixture warmedto 0° C. over 30 minutes. The reaction mixture was quenched withmethanol and Rochelle's salt (1M aqueous solution) and the aqueousextracted with ethyl acetate. The combined organic extracts were dried(Na₂SO₄) and concentrated in vacuo affording2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanol as an oil(156 mg, 95%). LCMS: R_(T)=2.60 min, [M+H]⁺=294/296/298.

Step 4:2-Chloro-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanol (145 mg,0.49 mmol) in THF (5 mL) was added sodium hydride (59 mg, 1.50 mmol, 60%dispersion in mineral oil) and the resulting mixture stirred at RT for30 minutes. The reaction mixture was quenched with saturated aqueousammonium chloride solution and extracted with ethyl acetate. Thecombined organic extracts were dried (Na₂SO₄) and concentrated in vacuoaffording2-Chloro-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine as awhite solid (114 mg, 90%). LCMS: R_(T)=2.62 min, [M+H]⁺=258/260.

Step 5: A mixture of2-chloro-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine (114mg, 0.44 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(196 mg, 0.88 mmol), PdCl₂dppf.DCM (36 mg, 0.04 mmol) and cesiumcarbonate (430 mg, 1.32 mmol) in dioxane (5 mL) and water (0.5 mL) wasdegassed and then heated at 100° C. for 2 hours. Further5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(100 mg, 0.44 mmol), PdCl₂dppf.DCM (36 mg, 0.04 mmol) were added and themixture heated at 100° C. for a further 2 hours. The reaction mixturewas diluted with water and extracted with ethyl acetate. The combinedorganic extracts were dried (Na₂SO₄) and concentrated in vacuo. Theresulting residue was loaded onto an Isolute® SCX-2 cartridge which waswashed with methanol and the product eluted with 2M ammonia in methanol.The basic fractions were combined and concentrated in vacuo. Theresulting residue was purified by column chromatography (SiO₂, gradient0-10% methanol in DCM) then triturated with diethyl ether affording 101(37 mg, 27%). LCMS: R_(T)=2.75 min, [M+H]⁺=317. ¹H NMR (400 MHz, DMSO):δ 8.90 (2H, s), 7.01 (2H, s), 4.42-4.36 (2H, m), 4.25-4.18 (2H, m), 3.70(8H, s).

Example 1025-[(6R)-6-methyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl]pyrimidin-2-amine102

Step 1: (R)-2-(2,4,6-Trichloro-pyrimidin-5-yloxy)-propionic acid ethylester

A mixture of 2,4,6-trichloro-pyrimidin-5-ol (600 mg, 3.01 mmol),(R)-2-hydroxy-propionic acid ethyl ester (411 μL, 3.60 mmol),triphenylphosphine (906 mg, 3.45 mmol) and DIAD (678 μL, 3.45 mmol) indioxane (5 mL) was stirred at RT for 18 hours before being concentratedin vacuo. The resulting residue was purified by column chromatography(SiO₂, gradient 0-25% ethyl acetate in cyclohexane) affording(R)-2-(2,4,6-Trichloro-pyrimidin-5-yloxy)-propionic acid ethyl ester(522 mg, 52%). ¹H NMR (400 MHz, CDCl₃): δ 4.30-4.16 (2H, m), 4.12 (1H,q, J=7.14 Hz), 1.70 (3H, d, J=6.78 Hz), 1.29 (3H, t, J=7.19 Hz).

Step 2:(R)-2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propionic acidethyl ester

A mixture of (R)-2-(2,4,6-trichloro-pyrimidin-5-yloxy)-propionic acidethyl ester (522 mg, 1.74 mmol), morpholine (152 μL, 1.74 mmol) andtriethylamine (484 μL, 3.48 mmol) in ethanol (15 mL) was stirred at RTfor 18 hours then partitioned between ethyl acetate and water. Theaqueous phase was extracted with further ethyl acetate and the combinedorganic extracts dried (Na₂SO₄) and concentrated in vacuo affording(R)-2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propionic acidethyl ester (490 mg, 80%). LCMS: R_(T)=3.57 min, [M+H]⁺=350/352.

Step 3:(R)-2-Chloro-6-methyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of(R)-2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propionic acidethyl ester (175 mg, 0.50 mmol) in THF (5 mL) at −78° C. was addedDIBAL-H (2.0 mL, 2.0 mmol, 1M solution in THF) and the resulting mixturewarmed to 0° C. over 30 minutes. The reaction mixture was quenched withRochelle's salt (1M aqueous solution) and the aqueous extracted withethyl acetate. The combined organic extracts were dried (Na₂SO₄) andconcentrated in vacuo. The resulting residue was taken up into THF (10mL), to the resultant solution was added sodium hydride (60 mg, 1.50mmol, 60% dispersion in mineral oil) and the resulting mixture stirredat RT for 18 hours. The reaction mixture was quenched with saturatedaqueous ammonium chloride solution and extracted with ethyl acetate. Thecombined organic extracts were dried (Na₂SO₄) and concentrated in vacuoaffording(R)-2-Chloro-6-methyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas a white solid (157 mg, quantitative). LCMS: R_(T)=2.89 min,[M+H]⁺=272/274.

Step 4: A mixture of(R)-2-chloro-6-methyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(65 mg, 0.24 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine (80mg, 0.36 mmol), Pd(OAc)₂ (1 mg, 10 mol %), (S)-phos (4.1, 20 mol %) andpotassium phosphate tribasic (106 mg, 0.50 mmol) in n-butanol (2.25 mL)and water (0.25 mL) was degassed and then heated at 100° C. for 18hours. The reaction mixture was diluted with water and extracted withethyl acetate. The combined organic extracts were dried (Na₂SO₄) andconcentrated in vacuo. The resulting residue was purified by columnchromatography (SiO₂, gradient 0-5% methanol in DCM) affording 102 (23mg, 29%). LCMS: R_(T)=3.09 min, [M+H]⁺=331. ¹H NMR (400 MHz, DMSO): δ9.15 (2H, s), 5.73 (2H, s), 4.44 (1H, dd, J=11.44, 2.17 Hz), 4.31-4.24(1H, m), 4.08 (1H, dd, J=11.45, 8.21 Hz), 3.89-3.73 (8H, m), 1.42 (3H,d, J=6.38 Hz).

Example 1035-(7-methyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine103

Step 1: (2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-acetic acid

To a solution of(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-acetic acid ethylester (581 mg, 1.73 mmol) in THF (10 mL) and water (2.5 mL) was addedLiOH (951 μL, 1.90 mmol, 2M aqueous solution) and the resulting mixturestirred at RT for 2.5 hours. The reaction mixture was quenched with 1MHCl and extracted with ethyl acetate. The combined organic extracts weredried (Na₂SO₄) and concentrated in vacuo affording(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-acetic acid as a whitesolid (515 mg, 97%). ¹H NMR (400 MHz, CDCl₃): δ 4.59 (2H, s), 3.93-3.87(4H, m), 3.81-3.75 (4H, m).

Step 2:2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-N-methoxy-N-ethyl-acetamide

A mixture of (2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-aceticacid (722 mg, 2.34 mmol) and CDI (493 mg, 3.04 mmol) in DCM (5 mL) wasstirred at RT for 1.5 hours before the addition of triethylamine (457μL, 3.28 mmol) and O,N-dimethyl-hydroxylamine hydrochloride (320 mg,3.28 mmol). The resulting mixture was stirred for 18 hours then quenchedwith 1M HCl. The aqueous layer was extracted with DCM and the combinedorganic extracts were washed with brine, then dried (Na₂SO₄) andconcentrated in vacuo to afford2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-N-methoxy-N-ethyl-acetamideas a yellow oil (830 mg, quantitative). LCMS: R_(T)=2.83 min,[M+H]⁺=351/353/355.

Step 3: 1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propan-2-one

To a solution of2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-N-methoxy-N-ethyl-acetamide(200 mg, 0.60 mmol) in THF (4 mL) at −78° C. was added methylmagnesiumbromide (1.28 mL, 1.79 mmol, 1.4 M solution in THF/toluene) and theresulting mixture warmed to RT and stirred for 3 hours. The resultingmixture was quenched with 1M HCl and extracted with ethyl acetate. Thecombined organic extracts were washed with brine, then dried (Na₂SO₄)and concentrated in vacuo affording1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propan-2-one as awhite solid (146 mg, 80%). LCMS: R_(T)=2.90 min, [M+H]⁺=306/308/310.

Step 4: 1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propan-2-ol

To a solution of1-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propan-2-one (146mg, 0.48 mmol) in THF (5 mL) at −78° C. was added DIBAL-H (580 μL, 0.58mmol, 1M solution in toluene) and the resulting mixture warmed to 0° C.and stirred for 1.5 hours. The reaction mixture was cooled to −78° C.before the addition of further DIBAL-H (480 μL, 0.48 mmol) and themixture warmed to 0° C. and stirred for 2 hours. The reaction mixturewas quenched with Rochelle's salt (1M aqueous solution) and the mixtureextracted with ethyl acetate. The combined organic extracts were washedwith brine, then dried (Na₂SO₄) and concentrated in vacuo affording1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propan-2-ol (148 mg,quantitative). LCMS: R_(T)=2.84 min, [M+H]⁺=308/310/312.

Step 5:2-Chloro-7-methyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of1-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-propan-2-ol (148 mg,0.48 mmol) in THF (8 mL) was added sodium hydride (58 mg, 1.44 mmol, 60%dispersion in mineral oil) and the resulting mixture stirred at RT for 4hours. The reaction mixture was quenched with saturated aqueous ammoniumchloride solution and extracted with ethyl acetate. The combined organicextracts were dried (Na₂SO₄) and concentrated in vacuo affording2-Chloro-7-methyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas an off-white solid (139 mg, quantitative). LCMS: R_(T)=2.94 min,[M+H]⁺=272/274.

Step 6: A mixture of2-chloro-7-methyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(99 mg, 0.36 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine (97mg, 0.44 mmol), Pd(PPh₃)₂Cl₂ (28 mg, 0.04 mmol) and sodium carbonate(1.2 mL, 1.20 mmol, 1M aqueous solution) in acetonitrile (1.2 mL) wasdegassed then heated at 140° C. for 25 minutes using microwaveirradiation. The reaction mixture was filtered, flushing with ethylacetate. The filtrate phases were separated and the aqueous layerextracted with ethyl acetate. The combined organic extracts were dried(Na₂SO₄) and concentrated in vacuo. The resulting residue was purifiedby column chromatography (SiO₂, gradient 0-4% methanol in DCM) thentriturated with diethyl ether affording 103 as a white solid (12 mg,11%). LCMS: R_(T)=3.06 min, [M+H]⁺=331. ¹H NMR (400 MHz, DMSO): δ 8.90(2H, s), 7.01 (2H, s), 4.57-4.47 (1H, m), 4.36 (1H, dd, J=11.41, 2.34Hz), 3.82 (1H, dd, J=11.43, 7.60 Hz), 3.77-3.61 (8H, m), 1.34 (3H, d,J=6.44 Hz)

Example 1045-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine104

Step 1:1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-2-methyl-propan-2-ol

To a solution of(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-acetic acid ethylester from Example 101 (2.45 g, 7.29 mmol) in THF (100 mL) at −78° C.was added methylmagnesium bromide (14.58 mL, 43.74 mmol, 3.0 M solutionin diethyl ether) and the resulting mixture stirred for 2 hours at 0° C.The reaction mixture was quenched with saturated ammonium chloridesolution and extracted with ethyl acetate. The combined organic extractswere dried (Na₂SO₄) and concentrated in vacuo affording1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-2-methyl-propan-2-olas an amber gum (2.18 g, 93%). LCMS: R_(T)=3.00 min, [M+H]⁺=322/324/326.

Step 2:2-Chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of1-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-2-methyl-propan-2-ol(2.18 g, 6.77 mmol) in THF (150 mL) was added sodium hydride (812 mg,20.31 mmol, 60% dispersion in mineral oil) and the resulting mixturestirred at RT for 5 hours. The reaction mixture was quenched withsaturated aqueous ammonium chloride solution and extracted with ethylacetate. The combined organic extracts were dried (Na₂SO₄) andconcentrated in vacuo. The resultant residue was triturated withcyclohexane affording2-Chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas a white solid (1.35 g, 70%). LCMS: R_(T)=3.03 min, [M+H]⁺=286/288.

Step 3:5-(7,7-Dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)-pyrimidin-2-ylamine

A mixture of2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(285 mg, 1.00 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(440 mg, 1.99 mmol), Pd(PPh₃)₂Cl₂ (70 mg, 0.10 mmol) and sodiumcarbonate (3.3 mL, 3.3 mmol, 1M aqueous solution) in acetonitrile (7 mL)was degassed then heated at 120° C. for 30 minutes using microwaveirradiation. The reaction mixture was diluted with methanol and DCM andabsorbed onto diatomaceous earth. The resulting residue was purified bycolumn chromatography (SiO₂, gradient 0-4% methanol in DCM then SiO₂,gradient 0-100% ethyl acetate in cyclohexane) affording 104 as a whitesolid (245 mg, 71%). LCMS: R_(T)=3.28 min, [M+H]⁺=345. ¹H NMR (400 MHz,DMSO): δ 8.90 (2H, s), 7.00 (2H, s), 3.97 (2H, s), 3.70 (8H, s), 1.35(6H, s).

Example 1055-[4-morpholino-7-(trifluoromethyl)-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl]pyrimidin-2-amine105

Step 1:2-Chloro-4-morpholin-4-yl-7-trifluoromethyl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

A microwave vial was charged with 2,4,6-trichloro-pyrimidin-5-ol (200mg, 1.00 mmol) and 2-trifluoromethyl-oxirane (429 μL, 5.01 mmol) thensealed. The reaction mixture was heated at 95° C. for 48 hours beforebeing cooled and concentrated in vacuo. The resultant residue wasdissolved into IMS (5 mL) then treated with triethylamine (198 μL, 1.42mmol) and morpholine (83 μL, 0.95 mmol). The orange mixture was stirredfor 18 hours at RT before being concentrated in vacuo. The resultingresidue was purified by column chromatography (SiO₂, gradient 0-20%ethyl acetate in cyclohexane) affording2-Chloro-4-morpholin-4-yl-7-trifluoromethyl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas a white solid (105 mg, 32%). LCMS: R_(T)=3.20 min, [M+H]⁺=326/328.

Step 2: A microwave vial was charged with2-chloro-4-morpholin-4-yl-7-trifluoromethyl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(105 mg, 0.32 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(143 mg, 0.64 mmol), Pd(PPh₃)₂Cl₂ (23 mg, 0.03 mmol) and sodiumcarbonate (1.06 mL, 1.06 mmol, 1M aqueous solution) in acetonitrile (1.6mL) before the vessel was evacuated and back filled with argon (×3). Themixture was heated at 120° C. for 30 minutes using microwaves. Thereaction mixture was diluted with H₂O/DCM/methanol then absorbed ontodiatomaceous earth before being purified by column chromatography (SiO₂,gradient 0-4% methanol in DCM then SiO₂ then gradient 0-90% ethylacetate in cyclohexane) followed by trituration in diethyl ether to give105 as a white solid (60 mg, 49%). LCMS: R_(T)=3.52 min, [M+H]⁺=385. ¹HNMR (400 MHz, DMSO): δ 8.92 (2H, s), 7.08 (2H, s), 5.51-5.41 (1H, m),4.53-4.41 (2H, m), 3.78-3.65 (8H, m)

Example 1065-(4-morpholinospiro[6H-[1,4]dioxino[2,3-d]pyrimidine-7,1′-cyclopropane]-2-yl)pyrimidin-2-amine106

Step 1:1-(2,6-Dichloro-5-methoxy-pyrimidin-4-yloxy)-cyclopropanecarboxylic acidmethyl ester

To a solution of 2,4,6-trichloro-5-methoxy-pyrimidine (213 mg, 1.00mmol) and 1-hydroxy-cyclopropanecarboxylic acid methyl ester (139 mg,1.20 mmol) in THF (4 mL) was added sodium hydride (48 mg, 1.20 mmol, 60%dispersion in mineral oil) at −78° C. The resulting mixture was allowedto warm to RT and stirred for 2 hours. The reaction mixture was quenchedwith saturated aqueous ammonium chloride solution and extracted withethyl acetate. The combined organic extracts were dried (Na₂SO₄) andconcentrated in vacuo affording1-(2,6-Dichloro-5-methoxy-pyrimidin-4-yloxy)-cyclopropanecarboxylic acidmethyl ester as a white solid (300 mg, quantitative). LCMS: R_(T)=3.49min, [M+H]⁺=341/343/345.

Step 2:[1-(2,6-Dichloro-5-methoxy-pyrimidin-4-yloxy)-cyclopropyl]-methanol

To a solution of1-(2,6-dichloro-5-methoxy-pyrimidin-4-yloxy)-cyclopropanecarboxylic acidmethyl ester (293 mg, 1.00 mmol) in DCM (8 mL) at −78° C. was addedDIBAL-H (4.00 mL, 4.00 mmol, 1M solution in THF) and the resultingmixture warmed to 0° C. and stirred for 90 minutes. The reaction mixturewas quenched with methanol followed by Rochelle's salt (1M aqueoussolution) and the mixture extracted with ethyl acetate. The combinedorganic extracts were dried (Na₂SO₄) and concentrated in vacuo affording[1-(2,6-Dichloro-5-methoxy-pyrimidin-4-yloxy)-cyclopropyl]-methanol asan oil (220 mg, 83%). LCMS: R_(T)=2.86 min, [M+H]⁺ 195/197/199.

Step 3: 2,4-Dichloro-6-(1-hydroxymethyl-cyclopropoxy)-pyrimidin-5-ol

A mixture of[1-(2,6-dichloro-5-methoxy-pyrimidin-4-yloxy)-cyclopropyl]-methanol (800mg, 3.02 mmol) and lithium chloride (600 mg, 14.15 mmol) in anhydrousDMF (3 mL) was heated at 140° C. for 10 minutes using microwaveirradiation, then concentrated in vacuo. The resulting residue waspurified by column chromatography (SiO₂, gradient 0-5% methanol in DCM)affording 2,4-Dichloro-6-(1-hydroxymethyl-cyclopropoxy)-pyrimidin-5-ol(300 mg, 40%). LCMS: R_(T)=2.52 min, [M−H]⁻=249/251/253.

Step 4:7-Cyclopropyl-2,4-dichloro-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of2,4-dichloro-6-(1-hydroxymethyl-cyclopropoxy)-pyrimidin-5-ol (135 mg,0.54 mmol) and triphenylphosphine (180 mg, 0.65 mmol) in THF (8 mL) wasadded DIAD (128 μL, 0.65 mmol) and the mixture stirred at RT for 30minutes, then concentrated in vacuo. The resulting residue was purifiedby column chromatography (SiO₂, gradient 0-25% ethyl acetate incyclohexane) affording7-Cyclopropyl-2,4-dichloro-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine asa white solid (90 mg, 72%). ¹H NMR (400 MHz, CDCl₃): δ 4.30 (2H, s),1.36-1.29 (2H, m), 1.01-0.95 (2H, m).

Step 5:2-Chloro-7-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

A mixture of7-cyclopropyl-2,4-dichloro-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine (93mg, 0.40 mmol), morpholine (150 μL, 1.71 mmol) and triethylamine (80 μL,0.57 mmol) in IMS (3 mL) was heated at 140° C. for 35 minutes usingmicrowave irradiation before being concentrated in vacuo. The resultingresidue was purified by column chromatography (SiO₂, 0-20% ethyl acetatein cyclohexane) affording2-Chloro-7-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas a white solid (98 mg, 87%). LCMS: R_(T)=3.02 min, [M+H]⁺=284/286.

Step 6: A mixture of2-chloro-7-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(98 mg, 0.34 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(144 mg, 0.65 mmol), Pd(PPh₃)₂Cl₂ (25 mg, 0.035 mmol) and sodiumcarbonate (1.0 mL, 1.0 mmol, 1M aqueous solution) in acetonitrile (4 mL)was degassed then heated at 120° C. for 30 minutes using microwaveirradiation. The reaction mixture was loaded onto an Isolute® SCX-2cartridge which was washed with methanol and the product eluted with 2Mammonia in methanol. The basic fractions were combined and concentratedin vacuo. The resulting residue was purified by reverse phase HPLC(Phenomenex Gemini 5 μm C18, 0.1% HCO₂H in water on a gradient ofacetonitrile 5-98%) affording 106 as a white solid (41 mg, 35%). LCMS:R_(T)=3.24 min, [M+H]⁺=343. ¹H NMR (400 MHz, CDCl₃): δ 9.12 (2H, s),5.20 (2H, broad s), 4.16 (2H, s), 3.82 (8H, s), 1.29 (2H, t, J=6.89 Hz),0.88 (2H, t, J=6.88 Hz).

Example 1075-(6-cyclopropyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine107

Step 1: Cyclopropyl-(2,4,6-trichloro-pyrimidin-5-yloxy)-acetic acidmethyl ester

To a solution of 2,4,6-trichloro-pyrimidin-5-ol (250 mg, 1.25 mmol),cyclopropyl-hydroxy-acetic acid methyl ester (245 mg, 1.88 mmol) andtriphenylphosphine (493 mg, 1.88 mmol) in THF (12 mL) was added DIAD(370 μL, 1.88 mmol) dropwise. The resultant suspension was stirred for18 hours before the addition of dioxane (10 mL) and the reaction stirredfor a further 6 hours. The heterogeneous mixture was concentrated invacuo and the resultant residue was purified by column chromatography(SiO₂, gradient 0-30% ethyl acetate in cyclohexane) affordingCyclopropyl-(2,4,6-trichloro-pyrimidin-5-yloxy)-acetic acid methyl esteras a white solid (224 mg, 32%). LCMS: R_(T)=3.66 min,[M+H]⁺=311/313/315.

Step 2:Cyclopropyl-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-aceticacid methyl ester

To a suspension ofcyclopropyl-(2,4,6-trichloro-pyrimidin-5-yloxy)-acetic acid methyl ester(224 mg, 0.72 mmol) in IMS (4 mL) was added triethylamine (150 μL, 1.08mmol) followed by morpholine (63 μL, 0.72 mmol). The yellow solution wasstirred for 2.5 hours at RT before being concentrated in vacuo. Theresultant residue was purified by column chromatography (SiO₂, gradient0-20% ethyl acetate in cyclohexane) affordingCyclopropyl-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-aceticacid methyl ester as a colourless gum (233 mg, 89%). ¹H NMR (CDCl₃): δ3.99 (1H, d, J=9.54 Hz), 4.03-3.83 (4H, m), 3.80-3.75 (4H, m), 3.78 (3H,s), 1.32-1.21 (1H, m), 0.70-0.57 (2H, m), 0.52-0.45 (1H, m), 0.33-0.26(1H, m).

Step 3:2-Cyclopropyl-2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanol

To a solution ofcyclopropyl-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-aceticacid methyl ester (233 mg, 0.64 mmol) in THF (6.5 mL) at −78° C. underan atmosphere of nitrogen was added DIBAL-H (1.93 mL, 1.93 mmol, 1.0M intoluene) dropwise. The reaction mixture was warmed to RT and stirred for20 hours before being quenched with Rochelle's salt (15 mL, 1M aqueoussolution). The mixture was extracted with ethyl acetate (3×30 mL) andthe combined organic phases were dried (Na₂SO₄) then concentrated invacuo to give2-Cyclopropyl-2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanolas a colorless gum (215 mg, quantitative). ¹H NMR (CDCl₃): δ 3.94-3.88(2H, m), 3.87-3.82 (4H, m), 3.79-3.73 (4H, m), 3.48-3.41 (1H, m),1.11-0.99 (1H, m), 0.60-0.51 (1H, m), 0.51-0.42 (1H, m), 0.19-0.11 (1H,m), 0.03-−0.05 (1H, m).

Step 4:2-Chloro-6-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of2-cyclopropyl-2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanol(215 mg, 0.64 mmol) in THF (10 mL) was added sodium hydride (71 mg, 1.93mmol, 65% dispersion in mineral oil) and the reaction stirred for 2hours. The reaction was quenched with saturated aqueous ammoniumchloride solution (15 mL) and water (5 mL) then extracted with ethylacetate (3×20 mL). The combined organic phases were washed with brine(15 mL), dried (Na₂SO₄) then concentrated in vacuo to give2-Chloro-6-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas a white solid (191 mg, quantitative). LCMS: R_(T)=3.18 min,[M+H]⁺=298/300.

Step 5: A microwave vial was charged with2-chloro-6-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(191 mg, 0.64 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(283 mg, 1.28 mmol), Pd(PPh₃)₂Cl₂ (45 mg, 0.06 mmol), sodium carbonate(2.11 mL, 2.11 mmol, 1M aqueous solution) and acetonitrile (2.5 mL). Thevessel was sealed then evacuated and refilled with argon (×3) beforebeing heated at 120° C. for 30 minutes using microwave irradiation. Thereaction mixture was filtered before being purified by columnchromatography (SiO₂, gradient 0-4% methanol in DCM). The resultantresidue was triturated with diethyl ether to give 107 as a white solid(21 mg, 9%). LCMS: R_(T)=3.54 min, [M+H]⁺=357. ¹H NMR (400 MHz,DMSO-d₆): δ 8.89 (2H, s), 7.00 (2H, s), 4.53 (1H, dd, J=11.48, 2.34 Hz),4.22 (1H, dd, J=11.50, 7.87 Hz), 3.75-3.67 (8H, m), 3.65-3.58 (1H, m),1.01-0.99 (1H, m), 0.63-0.55 (2H, m), 0.51-0.42 (2H, m).

Example 1085-(7-tert-butyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine108

Step 1: 3,3-Dimethyl-1-(2,4,6-trichloro-pyrimidin-5-yloxy)-butan-2-ol

A mixture of 2,4,6-trichloro-pyrimidin-5-ol (400 mg, 2.01 mmol) and2-tert-butyl oxirane (3 mL) was heated at 100° C. for 3 hours beforebeing cooled and concentrated in vacuo. The resultant residue wastriturated in hot methanol and the insoluble material removed. Thefiltrate was concentrated in vacuo and the resultant residue waspurified by column chromatography (SiO₂, 0-15% ethyl acetate incyclohexane) to give3,3-Dimethyl-1-(2,4,6-trichloro-pyrimidin-5-yloxy)-butan-2-ol as a whitesolid (275 mg, 52%). ¹H NMR (400 MHz, CDCl₃): δ 4.28 (1H, dd, J=9.38,2.19 Hz), 4.00 (1H, t, J=9.19 Hz), 3.76-3.69 (1H, m), 2.53 (1H, d,J=3.48 Hz), 0.95 (9H, s).

Step 2:1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-3,3-dimethyl-butan-2-ol

To a solution of3,3-dimethyl-1-(2,4,6-trichloro-pyrimidin-5-yloxy)-butan-2-ol (275 mg,1.05 mmol) in IMS (3 mL) was added triethylamine (291 μL, 2.09 mmol)followed by morpholine (100 μL, 1.15 mmol) and stirred at RT for 2hours. The reaction was quenched with saturated aqueous sodium hydrogencarbonate solution then extracted with ethyl acetate. The combinedorganic extracts were dried (Na₂SO₄) then concentrated in vacuo giving1-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-3,3-dimethyl-butan-2-ol(255 mg, 77%). LCMS: R_(T)=3.57 min, [M+H]⁺=350/352/354.

Step 3:7-tert-Butyl-2-chloro-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of1-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-3,3-dimethyl-butan-2-ol(255 mg, 0.73 mmol) in THF (10 mL) was added sodium hydride (87 mg, 2.18mmol, 60% dispersion in mineral oil) and the mixture was stirred at RTfor 1.5 hours. The reaction was quenched with saturated aqueous ammoniumchloride solution then extracted with ethyl acetate (×2). The combinedorganic layers were dried (Na₂SO₄) then passed through a pad of silicaeluting with ethyl acetate. The relevant fractions were concentrated invacuo giving7-tert-Butyl-2-chloro-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas a white solid (250 mg, 100%). LCMS: R_(T)=3.64 min, [M+H]⁺=314/316.

Step 4: A microwave vial was charged with7-tert-butyl-2-chloro-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(50 mg, 0.16 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine (70mg, 0.32 mmol) andbis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II)(11 mg, 0.02 mmol, 10 mol %) then sealed. The vessel was evacuated andback filled with nitrogen before the addition of sodium carbonate (0.25mL, 0.25 mmol, 1M aqueous solution) and acetonitrile (0.75 mL). Themixture was heated at 150° C. for 30 minutes using microwave irradiationbefore the mixture was filtered and the filtrate concentrated in vacuo.The resultant residue purified by column chromatography (SiO₂, 0-5%methanol in DCM) then triturated with cyclohexane/diethyl ether to give108 as a white solid (13 mg, 22%). LCMS: R_(T)=4.04 min, [M+H]⁺=373. ¹HNMR (400 MHz, CDCl₃): δ 9.14 (2H, s), 5.25 (2H, s), 4.44 (1H, dd,J=11.08, 1.92 Hz), 3.99 (1H, dd, J=8.97, 1.89 Hz), 3.91-3.69 (9H, m),1.12 (9H, s).

Example 1093-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)phenol109

A microwave vial was charged with2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidinefrom Example 104 (50 mg, 0.18 mmol),3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenol (58 mg, 0.26mmol), Pd(PPh₃)₂Cl₂ (12 mg, 0.02 mmol) and sodium carbonate (0.5 mL, 0.5mmol, 1M aqueous solution) in acetonitrile (1.5 mL) then evacuated andback filled with nitrogen before being heated at 120° C. for 30 minutesusing microwave irradiation. The reaction mixture was loaded onto anIsolute® SCX-2 cartridge which was washed with methanol and the producteluted with 2M ammonia in methanol. The basic fractions were combinedand concentrated in vacuo. The resulting residue was purified bytrituration with diethyl ether affording 109 as a white solid (38 mg,63%). LCMS: R_(T)=4.13 min, [M+H]⁺=344. ¹H NMR (400 MHz, CDCl₃): δ7.87-7.82 (2H, m), 7.26 (1H, t, J=7.6 Hz), 6.91 (1H, dd, J=2.4, 7.6 Hz),3.92 (2H, s), 3.90-3.80 (8H, m), 1.47 (6H, s).

Example 1102-(2-methoxypyrimidin-5-yl)-7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidine110

A microwave vial was charged with2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidinefrom Example 104 (50 mg, 0.18 mmol), 2-methoxypyrimidin-5-ylboronic acid(42 mg, 0.27 mmol), Pd(PPh₃)₂Cl₂ (12 mg, 0.02 mmol) and sodium carbonate(0.5 mL, 0.5 mmol, 1M aqueous solution) in acetonitrile (1.5 mL) thenevacuated and back filled with nitrogen before being heated at 120° C.using microwave irradiation. The reaction mixture was loaded onto anIsolute® SCX-2 cartridge which was washed with methanol and the producteluted with 2M ammonia in methanol. The basic fractions were combinedand concentrated in vacuo. The resulting residue was purified bytrituration with diethyl ether affording 110 as a white solid (30 mg,17%). LCMS: R_(T)=4.18 min, [M+H]⁺=360. ¹H NMR (400 MHz, CDCl₃): δ 9.33(2H, s), 4.07 (3H, s), 3.92 (2H, s), 3.83 (8H, s), 1.47 (6H, s)

Example 1115-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyridin-2-amine111

A microwave vial was charged with2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidinefrom Example 104 (50 mg, 0.18 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine (58mg, 0.26 mmol), Pd(PPh₃)₂Cl₂ (12 mg, 0.02 mmol) and sodium carbonate(0.5 mL, 0.5 mmol, 1M aqueous solution) in acetonitrile (1.5 mL) thenevacuated and back filled with nitrogen before being heated at 120° C.for 30 minutes using microwave irradiation. The reaction mixture wasloaded onto an Isolute® SCX-2 cartridge which was washed with methanoland the product eluted with 2M ammonia in methanol. The basic fractionswere combined and concentrated in vacuo. The resulting residue waspurified by reverse phase HPLC (Phenomenex Gemini 5 μm C18, 0.1% HCO₂Hin water on a gradient of acetonitrile 5-98%) affording 111 as a whitesolid (32 mg, 53%). LCMS: R_(T)=2.73 min, [M+H]⁺=344. ¹H NMR (400 MHz,CDCl₃): δ 9.01 (1H, d, J=2.1 Hz), 8.33 (1H, dd, J=2.1, 8.5 Hz), 6.50(1H, d, J=8.5 Hz), 4.61 (2H, broad s), 3.89 (2H, s), 3.86-3.77 (8H, m),1.43 (6H, s)

Example 112N-[4-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)phenyl]acetamide112

A microwave vial was charged with2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidinefrom Example 104 (50 mg, 0.18 mmol),N-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetamide(69 mg, 0.26 mmol), Pd(PPh₃)₂Cl₂ (12 mg, 0.02 mmol) and sodium carbonate(0.5 mL, 0.5 mmol, 1M aqueous solution) in acetonitrile (1.5 mL) thenwas evacuated and back filled with nitrogen before being heated at 120°C. for 30 minutes using microwave irradiation. The reaction mixture wasloaded onto an Isolute® SCX-2 cartridge which was washed with methanoland the product eluted with 2M ammonia in methanol. The basic fractionswere combined and concentrated in vacuo. The resulting residue waspurified by reverse phase HPLC (Phenomenex Gemini 5 μm C18, 0.1% HCO₂Hin water on a gradient of acetonitrile 5-98%) affording 112 as a whitesolid (40 mg, 59%). LCMS: R_(T)=2.73 min, [M+H]⁺=344. ¹H NMR (400 MHz,CDCl₃): δ 8.28 (2H, d, J=8.8 Hz), 7.54 (2H, d, J=8.8 Hz), 7.22 (1H,broad s), 3.90 (2H, s), 3.87-3.78 (8H, m), 2.19 (3H, s), 1.46 (6H, s)

Example 1135-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)-N-methyl-pyridin-2-amine113

A microwave vial was charged with2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidinefrom Example 104 (50 mg, 0.18 mmol),methyl-[5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl]-carbamicacid tert-butyl ester (88 mg, 0.26 mmol), Pd(PPh₃)₂Cl₂ (12 mg, 0.02mmol) and sodium carbonate (0.5 mL, 0.5 mmol, 1M aqueous solution) inacetonitrile (1.5 mL) then evacuated and back filled with nitrogenbefore being heated at 120° C. for 30 minutes using microwaveirradiation. The reaction mixture was loaded onto an Isolute® SCX-2cartridge which was washed with methanol and the product eluted with 2Mammonia in methanol. The basic fractions were combined and concentratedin vacuo. The resulting residue was purified by reverse phase HPLC(Phenomenex Gemini 5 μm C18, 0.1% HCO₂H in water on a gradient ofacetonitrile 5-98%) affording 113 as a white solid (20 mg, 32%). LCMS:R_(T)=2.79 min, [M+H]⁺=358. ¹H NMR (400 MHz, CDCl₃): δ 9.04 (1H, d,J=2.4 Hz), 8.35 (1H, dd, J=2.4, 8.6 Hz), 6.39 (1H, d, J=8.6 Hz), 4.84(1H, broad s), 3.89 (2H, s), 3.85-3.76 (8H, m), 2.97 (3H, d, J=5.2 Hz),1.45 (6H, s)

Example 114N-[3-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)phenyl]methanesulfonamide114

A microwave vial was charged with2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidinefrom Example 104 (50 mg, 0.18 mmol),3-(methanesulfonylamino)phenylboronic acid (57 mg, 0.27 mmol),Pd(PPh₃)₂Cl₂ (12 mg, 0.02 mmol) and sodium carbonate (0.5 mL, 0.5 mmol,1M aqueous solution) in acetonitrile (1.5 mL) then evacuated and backfilled with nitrogen before being heated at 120° C. for 30 minutes usingmicrowave irradiation. The reaction mixture was loaded onto an Isolute®SCX-2 cartridge which was washed with methanol and the product elutedwith 2M ammonia in methanol. The basic fractions were combined andconcentrated in vacuo. The resulting residue was purified by reversephase HPLC (Phenomenex Gemini 5 μm C18, 0.1% HCO₂H in water on agradient of acetonitrile 5-98%) affording 114 as a white solid (11 mg,15%). LCMS: R_(T)=4.22 min, [M+H]⁺=421. ¹H NMR (400 MHz, CDCl₃): δ8.19-8.12 (1H, m), 8.05-8.02 (1H, m), 7.44-7.37 (2H, m), 6.34 (1H, broads), 3.92 (2H, s), 3.88-3.79 (8H, m), 3.00 (3H, s), 1.47 (6H, s)

Example 1152-(1H-indol-5-yl)-7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidine115

A microwave vial was charged with2-chloro-7,7-dimethyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidinefrom Example 104 (50 mg, 0.18 mmol), 5-indolylboronic acid (42 mg, 0.26mmol), Pd(PPh₃)₂Cl₂ (12 mg, 0.02 mmol) and sodium carbonate (0.5 mL, 0.5mmol, 1M aqueous solution) in acetonitrile (1.5 mL) then evacuated andback filled with nitrogen before being heated to 120° C. for 30 minutesusing microwave irradiation. The reaction mixture was loaded onto anIsolute® SCX-2 cartridge which was washed with methanol and the producteluted with 2M ammonia in methanol. The basic fractions were combinedand concentrated in vacuo. The resulting residue was purified by reversephase HPLC (Phenomenex Gemini 5 μm C18, 0.1% HCO₂H in water on agradient of acetonitrile 5-98%) affording 115 as a white solid (11 mg,17%). LCMS: R_(T)=4.37 min, [M+H]⁺=367. ¹H NMR (400 MHz, CDCl₃): δ 8.67(1H, s), 8.28-8.20 (2H, m), 7.39 (1H, d, J=8.6 Hz), 7.23-7.19 (1H, m),6.61 (1H, broad s), 3.91 (2H, s), 3.90-3.82 (8H, m), 1.48 (6H, s)

Example 1185-(7-cyclopropyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine118

Step 1: (tert-Butyl-dimethyl-silanyloxy)-cyclopropyl-acetic acid methylester

To a solution of cyclopropyl-hydroxy-acetic acid methyl ester (1.00 g,7.68 mmol) in DCM (9 mL) was added tert-butyldimethylsilyl chloride(1.27 g, 8.45 mmol) and imidazole (628 mg, 9.22 mmol). The reactionmixture was stirred at RT for 65 hours before being quenched with water(10 mL) and 1M HCl (5 mL). The mixture was extracted with ethyl acetate(3×15 mL) and the combined organic phases were washed with 1M HCl (10mL), brine (10 mL), dried (Na₂SO₄) then concentrated in vacuo. This gave(tert-Butyl-dimethyl-silanyloxy)-cyclopropyl-acetic acid methyl ester asan amber oil (1.66 g, 88%). ¹H NMR (400 MHz, CDCl₃): δ 3.81 (1H, d,J=6.33 Hz), 3.74 (3H, s), 1.23-1.13 (1H, m), 0.89 (9H, s), 0.52-0.0.38(4H, m), 0.06 (3H, s), 0.04 (3H, s).

Step 2: 2-(tert-Butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethanol

To a solution of (tert-butyl-dimethyl-silanyloxy)-cyclopropyl-aceticacid methyl ester (1.66 g, 6.79 mmol) in DCM (27 mL) at −78° C. under anitrogen atmosphere was added DIBAL-H (20.37 mL, 20.37 mmol, 1.0M intoluene) dropwise. The reaction mixture was stirred at −78° C. for 2.5hours then at 0° C. for 1 hour before being quenched with Rochelle'ssalt (30 mL, 1M aqueous solution). The mixture was extracted with ethylacetate (3×30 mL) before the combined organic phases were dried (Na₂SO₄)and concentrated in vacuo giving2-(tert-Butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethanol as a paleorange oil (764 mg, 52%). ¹H NMR (400 MHz, CDCl₃): δ 3.66-3.49 (2H, m),3.09 (1H, ddd, J=7.96, 6.40, 3.80 Hz), 1.96 (1H, dd, J=7.43, 5.46 Hz),0.91 (9H, s), 0.91-0.82 (1H, m), 0.57-0.43 (2H, m), 0.34-0.24 (1H, m),0.23-0.14 (1H, m), 0.11 (3H, s), 0.07 (3H, s).

Step 3:5-[2-(tert-Butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethoxy]-2,4,6-trichloro-pyrimidine

To a solution of 2,4,6-trichloro-pyrimidin-5-ol (250 mg, 1.25 mmol),2-(tert-butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethanol (407 mg, 1.88mmol) and triphenylphosphine (493 mg, 1.88 mmol) in THF (12 mL) wasadded DIAD (370 μL, 1.88 mmol). The reaction mixture was stirred at RTfor 18 hours before being concentrated in vacuo. The resultant residuewas purified by column chromatography (SiO₂, gradient 0-30% ethylacetate in cyclohexane) to give5-[2-(tert-Butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethoxy]-2,4,6-trichloro-pyrimidineas a viscous yellow oil (411 mg, 83%). ¹H NMR (400 MHz, CDCl₃): δ 4.14(1H, dd, J=8.83, 6.02 Hz), 3.99 (1H, dd, J=8.82, 4.21 Hz), 3.60-3.53(1H, m), 1.13-1.03 (1H, m), 0.89 (9H, s), 0.56-0.50 (2H, m), 0.46-0.28(2H, m), 0.10 (3H, s), 0.07 (3H, s).

Step 4:4-{5-[2-(tert-Butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethoxy]-2,6-dichloro-pyrimidin-4-yl}-morpholine

To a solution of5-[2-(tert-butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethoxy]-2,4,6-trichloro-pyrimidine(411 mg, 1.03 mmol) in IMS (5 mL) was added triethylamine (215 μL, 1.55mmol) followed by morpholine (94 μL, 1.08 mmol). The reaction mixturewas stirred at RT for 4 hours before being concentrated in vacuo. Theresultant residue was purified by column chromatography (SiO₂, gradient0-20% ethyl acetate in cyclohexane) to afford4-{5-[2-(tert-Butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethoxy]-2,6-dichloro-pyrimidin-4-yl}-morpholineas a colorless gum (387 mg, 84%). LCMS: R_(T)=5.22 min,[M+H]⁺=448/450/452.

Step 5:1-Cyclopropyl-2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanol

To a solution of4-{5-[2-(tert-butyl-dimethyl-silanyloxy)-2-cyclopropyl-ethoxy]-2,6-dichloro-pyrimidin-4-yl}-morpholine(386 mg, 0.86 mmol) in THF (10 mL) was added TBAF (947 μL, 0.95 mmol,1.0 M solution in THF) dropwise. The reaction mixture was stirred at RTfor 3 hours before the reaction was quenched with saturated aqueousammonium chloride solution (20 mL). The mixture was extracted with ethylacetate (3×20 mL) and then combined organic phases were washed withbrine (10 mL), dried (Na₂SO₄) and concentrated in vacuo. The resultantresidue was purified by column chromatography (SiO₂, gradient 0-50%ethyl acetate) giving1-Cyclopropyl-2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanolas a colourless gum (196 mg, 68%). LCMS: R_(T)=3.04 min, [M+H]⁺=334/336.

Step 6:2-Chloro-7-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine

To a solution of1-cyclopropyl-2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethanol(196 mg, 0.59 mmol) in THF (10 mL) was added sodium hydride (65 mg, 1.77mmol, 65% dispersion in mineral oil) and the reaction mixture stirred atRT for 4 hours. The reaction was quenched with saturated aqueousammonium chloride solution (15 mL) then extracted with ethyl acetate(3×20 mL). The combined organic phases were dried (Na₂SO₄) andconcentrated in vacuo giving2-Chloro-7-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidineas a white solid (205 mg, quantitative). LCMS: R_(T)=3.19 min,[M+H]⁺=298/300.

Step 7: A microwave vial was charged with2-chloro-7-cyclopropyl-4-morpholin-4-yl-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidine(205 mg, 0.49 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(324 mg, 1.47 mmol), Pd(PPh₃)₂Cl₂ (51 mg, 0.07 mmol), sodium carbonate(2.41 mL, 2.41 mmol, 1M aqueous solution) and acetonitrile (2.5 mL). Thevessel was sealed then evacuated and back filled with argon (×3) beforebeing heated at 120° C. for 30 minutes using microwave irradiation. Thereaction mixture was filtered and the filtrate concentrated in vacuo.The resultant residue was purified by column chromatography (SiO₂,gradient 0-4% methanol in DCM), then triturated in diethylether/pentane. The resulting residue was purified by reverse phase HPLC(Phenomenex Gemini 5 μm C18, 0.1% HCO₂H in water on a gradient methanol10-98%) affording 118 as an off white solid (4.5 mg, 3%). LCMS:R_(T)=3.52 min, [M+H]⁺=357. ¹H NMR (400 MHz, DMSO-d₆): δ 8.91 (2H, s),7.01 (2H, s), 4.40 (1H, dd, J=11.38, 2.41 Hz), 4.01 (1H, dd, J=11.41,7.52 Hz), 3.80-3.60 (9H, m), 1.11-0.98 (1H, m), 0.69-0.41 (4H, m).

Example 1195-(8-methyl-4-morpholino-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-2-yl)pyrimidin-2-amine119

Step 1: Methyl-[2-(2,4,6-trichloro-pyrimidin-5-yloxy)-ethyl]-carbamicacid tert-butyl ester

A mixture of 2,4,6-trichloro-pyrimidin-5-ol (300 mg, 1.50 mmol),(2-hydroxy-ethyl)-methyl-carbamic acid tert-butyl ester (300 mg, 1.71mmol), triphenylphosphine (455 mg, 1.73 mmol) and DIAD (339 μL, 1.73mmol) in dioxane (2.5 mL) was stirred at RT for 1.5 hours thenconcentrated in vacuo. The resulting residue was purified by columnchromatography (SiO₂, gradient 0-50% ethyl acetate in cyclohexane)affording Methyl-[2-(2,4,6-trichloro-pyrimidin-5-yloxy)-ethyl]-carbamicacid tert-butyl ester (666 mg, quantitative). LCMS: R_(T)=3.09 min,[M-Boc+Na]⁺=279.

Step 2:[2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethyl]-methyl-carbamicacid tert-butyl ester

A mixture ofmethyl-[2-(2,4,6-trichloro-pyrimidin-5-yloxy)-ethyl]-carbamic acidtert-butyl ester (1.50 mmol), morpholine (130 μL, 1.50 mmol) andtriethylamine (417 μL, 3.00 mmol) in ethanol (5 mL) was stirred at RTfor 1 hour then partitioned between ethyl acetate and water. The aqueousphase was extracted with further ethyl acetate and the combined organicextracts dried (Na₂SO₄) and concentrated in vacuo affording[2-(2,4-Dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethyl]-methyl-carbamicacid tert-butyl ester (quantitative). LCMS: R_(T)=3.89 min,[M-CH₂CH₂NMeBoc+H]⁺=250/252/254.

Step 3:2-Chloro-8-methyl-4-morpholin-4-yl-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazine

A mixture of[2-(2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-ethyl]-methyl-carbamicacid tert-butyl ester (1.50 mmol) and 4M HCl in methanol (10 mL) wasstirred at RT for 1.5 hours then concentrated in vacuo and azeotropedwith ethanol. The resultant residue was dissolved in methanol (10 mL)then treated with triethylamine (3.0 mL) and the mixture stirred at RTfor 30 minutes before being concentrated in vacuo. The resulting residuewas purified by column chromatography (SiO₂, gradient 0-50% ethylacetate in cyclohexane) affording2-Chloro-8-methyl-4-morpholin-4-yl-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazine(368 mg, 91% over 3 steps). LCMS: R_(T)=2.94 min, [M+H]⁺=271/273.

Step 4: A mixture of2-chloro-8-methyl-4-morpholin-4-yl-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazine(120 mg, 0.44 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(196 mg, 0.88 mmol), PdCl₂dppf.DCM (36 mg, 0.04 mmol) and cesiumcarbonate (573 mg, 1.76 mmol) in dioxane (5 mL) and water (0.5 mL) wasdegassed and heated at 100° C. for 18 hours. The reaction mixture wasdiluted with water and extracted with ethyl acetate. The combinedorganic extracts were dried (Na₂SO₄) and concentrated in vacuo. Theresulting residue was loaded onto an Isolute® SCX-2 cartridge which waswashed with methanol and the product eluted with 2M ammonia in methanol.The basic fractions were combined and concentrated in vacuo. Theresulting residue was purified by column chromatography (SiO₂, gradient0-5% methanol in DCM) then triturated with diethyl ether affording 119(357 mg, 24%). LCMS: R_(T)=3.04 min, [M+H]⁺=330. ¹H NMR (400 MHz, DMSO):δ 8.94 (2H, s), 6.92 (2H, s), 4.16 (2H, t, J=4.37 Hz), 3.67 (4H, t,J=4.57 Hz), 3.52 (4H, t, J=4.56 Hz), 3.48 (2H, t, J=4.36 Hz), 3.13 (3H,s).

Example 1202-(2-aminopyrimidin-5-yl)-8-methyl-4-morpholino-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one120

Step 1:2-Chloro-8-methyl-4-morpholin-4-yl-8H-pyrimido[5,4-b][1,4]oxazin-7-one

A mixture of (2,4-dichloro-6-morpholin-4-yl-pyrimidin-5-yloxy)-aceticacid ethyl ester (501 mg, 1.49 mmol), methylamine (1.0 mL, 2.0 mmol, 2 Min THF) and triethylamine (250 μL, 1.79 mmol) in IMS (10 mL) was heatedat 140° C. for 35 minutes using microwave irradiation. The reactionmixture was concentrated in vacuo and the resulting residue purified bycolumn chromatography (SiO₂, 0-20% ethyl acetate in cyclohexane)affording2-Chloro-8-methyl-4-morpholin-4-yl-8H-pyrimido[5,4-b][1,4]oxazin-7-one(159 mg, 38%). LCMS: R_(T)=2.93 min, [M+H]⁺=285/287.

Step 2: A mixture of2-chloro-8-methyl-4-morpholin-4-yl-8H-pyrimido[5,4-b][1,4]oxazin-7-one(100 mg, 0.35 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine(116 mg, 0.53 mmol), Pd(PPh₃)₂Cl₂ (25 mg, 0.035 mmol) and sodiumcarbonate (1.0 mL, 1.0 mmol, 1M aqueous solution) in acetonitrile (4 mL)was degassed then heated at 120° C. for 30 minutes using microwaveirradiation. The reaction mixture was loaded onto an Isolute® SCX-2cartridge which was washed with methanol and the product eluted with 2Mammonia in methanol. The basic fractions were combined and concentratedin vacuo. The resulting residue was purified by reverse phase HPLC(Phenomenex Gemini 5 μm C18, 0.1% HCO₂H in water on a gradient ofacetonitrile 5-98%) affording 120 as a white solid (86 mg, 71%). LCMS:R_(T)=3.04 min, [M+H]⁺=344. ¹H NMR (400 MHz, CDCl₃): δ 9.14 (2H, s),5.25 (2H, broad s), 4.62 (2H, s), 3.81 (8H, s), 3.49 (3H, s).

Example 901 p110α (alpha) PI3K Binding Assay

Binding Assays Initial polarization experiments were performed on anAnalyst HT® 96-384 (Molecular Devices Corp, Sunnyvale, Calif.). Samplesfor fluorescence polarization affinity measurements were prepared byaddition of 1:3 serial dilutions of p110 alpha PI3K (Upstate CellSignaling Solutions, Charlottesville, Va.) starting at a finalconcentration of 20 ug/mL in polarization buffer (10 mM tris pH 7.5, 50mM NaCl, 4 mM MgCl₂, 0.05% Chaps, and 1 mM DTT) to 10 mM PIP₂(Echelon-Inc., Salt Lake City, Utah.) final concentration. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah.) 100 nM and 5 nM final concentrationsrespectively. Read with standard cut-off filters for the rhodaminefluorophore (λex=530 nm; λem=590 nm) in 384-well black low volumeProxiplates® (PerkinElmer, Wellesley, Mass.) Fluorescence polarizationvalues were plotted as a function of the protein concentration, and theEC₅₀ values were obtained by fitting the data to a 4-parameter equationusing KaleidaGraph® software (Synergy software, Reading, Pa.). Thisexperiment also establishes the appropriate protein concentration to usein subsequent competition experiments with inhibitors.

Inhibitor IC₅₀ values were determined by addition of the 0.04 mg/mL p110alpha PI3K (final concentration) combined with PIP₂ (10 mM finalconcentration) to wells containing 1:3 serial dilutions of theantagonists in a final concentration of 25 mM ATP (Cell SignalingTechnology, Inc., Danvers, Mass.) in the polarization buffer. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah.) 100 nM and 5 nM final concentrationsrespectively. Read with standard cut-off filters for the rhodaminefluorophore (λex=530 nm; λem=590 nm) in 384-well black low volumePROXIPLATES® (PerkinElmer, Wellesley, Mass.) Fluorescence polarizationvalues were plotted as a function of the antagonist concentration, andthe IC₅₀ values were obtained by fitting the data to a 4-parameterequation in Assay Explorer® software (MDL, San Ramon, Calif.).

Alternatively, inhibition of PI3K was determined in a radiometric assayusing purified, recombinant enzyme and ATP at a concentration of 1 μM.The Formula I compound was serially diluted in 100% DMSO. The kinasereaction was incubated for 1 hr at room temperature, and the reactionwas terminated by the addition of PBS. IC₅₀ values were subsequentlydetermined using sigmoidal dose-response curve fit (variable slope).

Example 902 In Vitro Cell Proliferation Assay

Efficacy of Formula I compounds was measured by a cell proliferationassay employing the following protocol (Promega Corp. Technical BulletinTB288; Mendoza et al (2002) Cancer Res. 62:5485-5488):

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells (PC3(prostate cancer), Detroit562 (pharynx carcinoma), or MDAMB361.1 (breastcancer) in medium was deposited in each well of a 384-well,opaque-walled plate.

2. Control wells were prepared containing medium and without cells.

3. The compound was added to the experimental wells and incubated for3-5 days.

4. The plates were equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo® Reagent equal to the volume of cellculture medium present in each well was added.

6. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

Alternatively, cells were seeded at optimal density in a 96 well plateand incubated for 4 days in the presence of test compound. Alamar Blue™was subsequently added to the assay medium, and cells were incubated for6 hr before reading at 544 nm excitation, 590 nm emission. EC₅₀ valueswere calculated using a sigmoidal dose response curve fit. The term EC₅₀refers to the half maximal effective concentration and is theconcentration at which a drug induces a response halfway between thebaseline and maximum after some specified exposure time. It is commonlyused as a measure of drug potency.

The anti-proliferative effects of Formula I exemplary compounds weremeasured by the CellTiter-Glo® Assay against various tumor cell lines,including the following:

Cell line: MDA-MB-361.1 Cell line: PC3 Tissue type: breast Tissue type:prostate Mutation Status: PI3K Mutation Status: PTEN No. EC50 (μmole)EC50 (μmole) 101 1.1  1.0  102 0.37  0.557 103 0.63  0.606 104 0.5770.406 105 0.275 0.50  106 0.544 0.489 107 0.961 1.2  108 0.213 0.338 1090.445 0.182 110 4.1  4.8  111 0.545 0.48  112 1.8  2.1  113 2    2.1 114 7.5+  10+    115 1.8  2.3  116 0.73  0.76  117 0.669 0.759 119 2.6 3.1  120 0.57  0.589

Example 903 Caco-2 Permeability

Caco-2 cells are seeded onto Millipore Multiscreen® plates at 1×10⁵cells/cm², and cultured for 20 days. Assessment of compound permeabilityis subsequently conducted. The compounds are applied to the apicalsurface (A) of cell monolayers and compound permeation into thebasolateral (B) compartment is measured. This is performed in thereverse direction (B-A) to investigate active transport. A permeabilitycoefficient value, P_(app), for each compound, a measure of the rate ofpermeation of the compound across the membrane, is calculated. Compoundsare grouped into low (P_(app)</=1.0×10⁶ cm/s) or high (P_(app)>/=1.0×10⁶cm/s) absorption potential based on comparison with control compoundswith established human absorption.

For assessment of a compound's ability to undergo active efflux, theratio of basolateral (B) to apical (A) transport compared with A to Bwas determined. Values of B−A/A−B>/=1.0 indicate the occurrence ofactive cellular efflux.

Example 904 Hepatocyte Clearance

Suspensions of cryopreserved human hepatocytes are used. Incubations areperformed at compound concentration of 1 mM or 3 μM at a cell density of0.5×10⁶ viable cells/mL. The final DMSO concentration in the incubationis about 0.25%. Control incubations are also performed in the absence ofcells to reveal any non-enzymatic degradation. Duplicate samples (50 μL)are removed from the incubation mixture at 0, 5, 10, 20, 40 and 60minutes (control sample at 60 minutes only) and added to methanolcontaining internal standard (100 μL)—to terminate the reaction.Tolbutamide, 7-hydroxycoumarin, and testosterone may be used as controlcompounds. Samples are centrifuged and the supernatants at each timepoint pooled for analysis by LC-MSMS. From a plot of ln peak area ratio(parent compound peak area/internal standard peak area) against time,intrinsic clearance (C_(int)) is calculated as follows: CL_(int)(μl/min/million cells)=V×k, where k is the elimination rate constant,obtained from the gradient of ln concentration plotted against time; Vis a volume term derived from the incubation volume and is expressed asuL 10⁶ cells⁻¹.

Example 905 Cytochrome P450 Inhibition

Formula I compounds may be screened against CYP450 targets (1A2, 2C9,2C19, 2D6, 3A4) at about 10 concentrations in duplicate, with a topconcentration of about 100 μM (one hundred micromolar). Standardinhibitors (furafylline, sulfaphenazole, tranylcypromine, quinidine,ketoconazole) may be used as controls. Plates may be read using a BMGLabTechnologies PolarStar™ in fluorescence mode.

Example 906 Cytochrome P450 Induction

Freshly isolated human hepatocytes from a single donor may be culturedfor about 48 hr prior to addition of Formula I compound at threeconcentrations and incubated for 72 hr. Probe substrates for CYP3A4 andCYP1A2 are added for 30 minutes and 1 hr before the end of theincubation. At 72 hr, cells and media are removed and the extent ofmetabolism of each probe substrate quantified by LC-MS/MS. Theexperiment is controlled by using inducers of the individual P450sincubated at one concentration in triplicate.

Example 907 Plasma Protein Binding

Solutions of Formula I compound (5 μm (five micromolar), 0.5% final DMSOconcentration) are prepared in buffer and 10% plasma (v/v in buffer). A96 well HT dialysis plate is assembled so that each well is divided intwo by a semi-permeable cellulose membrane. The buffer solution is addedto one side of the membrane and the plasma solution to the other side;incubations are then conducted at 37° C. over 2 hr in triplicate. Thecells are subsequently emptied, and the solutions for each batch ofcompounds are combined into two groups (plasma-free andplasma-containing) then analyzed by LC-MSMS using two sets ofcalibration standards for plasma-free (6 points) and plasma-containingsolutions (7 points). The fraction unbound value for the compound iscalculated.

Example 908 hERG Channel Blockage

Formula I compounds are evaluated for ability to modulate rubidiumefflux from HEK-294 cells stably expressing hERG potassium channelsusing established flux methodology. Cells are prepared in mediumcontaining RbCl, plated into 96-well plates and grown overnight to formmonolayers. The efflux experiment is initiated by aspirating the mediaand washing each well with 3×100 μL of pre-incubation buffer (containinglow [K⁺]) at room temperature. Following the final aspiration, 50 μL ofworking stock (2×) compound is added to each well and incubated at roomtemperature for 10 minutes. Stimulation buffer 50 μL (containing high[K+]) is then added to each well giving the final test compoundconcentrations. Cell plates are then incubated at room temperature for afurther 10 minutes. Supernatant 80 μL from each well is then transferredto equivalent wells of a 96-well plate and analyzed via atomic emissionspectroscopy. The compound is screened as 10 pt duplicate IC₅₀ curves,n=2, from a top concentration of 100 μM.

Example 909 In Vivo Tumor Xenograft

NCR nude mice (Taconic Farms, Ind.) were inoculated subcutaneously inthe right lateral thorax with 5 million U-87 MG Merchant (an in-housevariant derived from U-87 MG cells from ATCC, Manassas, Va.) cells inHBSS/Matrigel (1:1, v/v). Mice bearing tumor xenografts were dosed dailyorally by gavage for <28 days with drug or vehicle after being separatedinto different dose groups of similarly sized tumors. Tumor sizes wererecorded at least twice weekly over the course of the study. Mouse bodyweights were also recorded at least twice weekly, and the mice wereobserved daily. Tumor volume was measured in two perpendiculardimensions (length and width) using Ultra Cal-IV calipers (Model54-10-111; Fred V. Fowler Co., Inc.; Newton, Mass.) and analyzed usingExcel v.11.2 (Microsoft Corporation; Redmond, Wash.). Tumor inhibitiongraphs were plotted using GraphPad Prism™, Version 5.0c (GraphPadSoftware, Inc.; La Jolla, Calif.). The tumor volume was calculated withformula: Tumor size (mm³)=(longer measurement×shorter measurement²)×0.5

Animal body weights were measured using an Adventurer Pro™ AV812 scale(Ohaus Corporation; Pine Brook, N.J.). Graphs were generated usingGraphPad Prism™, Version 5.0c. Percent weight change was calculatedusing formula: individual percent weight change=((new weight/initialweight)−1)×100.

Mice whose tumor volume exceeded 2000 mm³ or whose body weight lossexceeded 20% of their starting weight were euthanized according toregulatory guidance.

The percent tumor growth inhibition (% TGI) at the end of study (EOS)was calculated using formula:

% TGI=(1−[(AUC/Day)_(Treatment)÷(AUC/Day)_(Control)])×100, where AUC/Dayis the area under the fitted tumor growth curve on the natural scaledivided by the number of study days. Log 2(tumor volume) growth traceswere fitted to each dose group with restricted cubic splines for thefixed time and dose effect in each group. Fitting was done via a linearmixed effects model, using the R package ‘nlme’, version 3.1-97 (11) inR version 2.12.0 (R Development Core Team 2008; R Foundation forStatistical Computing; Vienna, Austria).

A partial response (PR) was defined as a >50% reduction in startingtumor volume that never became a complete response (CR) at any day ofthe study. CR was defined as a 100% reduction in starting tumor volumeat any day of the study. Study tumor incidence (STI) reflected thenumber of animals in a group with a measureable tumor for their lasttumor volume measurement.

Linear mixed effect analysis was also employed to model the percentchange in body weight over time and in response to dose.

Example 910 Phospho AKT Induction Assay

In a 6-well tissue culture plate cells were seeded at 5×10⁵ cells perwell overnight. Cells were treated with an EC₈₀ of the Formula Icompound. Following treatment, cells were washed once with cold PBS andlysed in 1× Cell Extraction Buffer from Biosource (Carlsbad, Calif.)supplemented with protease inhibitors (Roche, Mannheim, Germany), 1 mMPMSF, and Phosphatase Inhibitor Cocktails 1 and 2 from Sigma (St. Louis,Mo.). Determination of protein concentration was performed using thePierce BCA Protein Assay Kit (Rockford, Ill.). Levels of pAkt (Ser⁴⁷³)and total Akt were assessed using bead kits from Biosource (Carlsbad,Calif.) and the Luminex™ Bio-Plex system (Bio-Rad, Hercules, Calif.).

Example 911 Blood-Brain Barrier Activity/Penetrant Assay MDCKI-MDR1 andMDCKII-Bcrp1 Assays

Madin-Darby canine kidney (MDCK) cells heterologously expressing eitherhuman Pgp or mouse Bcrp1 were used to determine whether the compoundswere substrate of these transporters, and thus assess the potential forblood-brain barrier permeation. MDR1-MDCKI cells were licensed from theNCI (National Cancer Institute, Bethesda, Md.) while Bcrp1-MDCKII cellswere obtained from the Netherlands Cancer Institutes (Amsterdam, TheNetherlands). Cells were seeded on 24-well Millipore filter plates 4days prior to use (polyester membrane, 1 μM pore size; Millipore;Billerica, Mass.) at a seeding density of 1.3×10⁵ cells/mL. Compoundswere tested at 5 μM in the apical to basolateral (A−B) and basolateralto apical (B−A) directions. The compounds were dissolved in transportbuffer consisting of Hank's balanced salt solution (HBSS) with 10 mMHEPES (Invitrogen Corporation, Grand Island, N.Y.). Lucifer Yellow(Sigma-Aldrich, St. Louis, Mo.) was used as the paracellular marker. Theapparent permeability (P_(app)) in the A−B and B−A directions wascalculated after a 2-hour incubation using the following equation:P _(app)=(dQ/dt)×1/C ₀×1/A

where dQ/dt is the rate of compound appearance in the receivercompartment, C₀ is the concentration in the donor compartment and A isthe surface area of the insert. The Efflux Ratio, defined asP_(app (B-A))/P_(app A-B)), was used to assess the extent of activeefflux undergone by the compounds with the transporter tested(P-glycoprotein or bcrp1). The compounds were analyzed by LC-MS/MS.

Brain protein binding in rats was measured for Formula I exemplarycompounds, including the following:

Brain protein binding No. (mean bound %) 101 71.5 102 80 105 81.7 10686.6 116 83 120 84

Example 912 Determination of Compound Concentration in the Brain

Brains were collected at 1 and 6 hours post-dose from 3 differentanimals at each time point, rinsed with ice-cold saline, weighed andstore at −80° C. until analysis. For compound quantitation, mouse brainswere homogenized in 3 volumes of water. The homogenates were extractedby protein precipitation with acetonitrile containing the internalstandard. LC-MS/MS analysis was conducted. Brain homogenatesconcentrations were converted to brain concentrations for thecalculations of brain-to-plasma ratios.

Example 913 Measurement of the Modulation of the PI3K Pathway in theBrain

For analysis of PI3K pathway modulation, cell extraction buffer(Invitrogen, Camarillo, Calif.) containing 10 mM Tris pH 7.4, 100 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1%Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate wassupplemented with phosphatase, protease inhibitors (Sigma, St. Louis,Mo.) and 1 mM PMSF and added to frozen brain biopsies. Brains collectedat 1 and 6 hrs post-dose were homogenized with a small pestle (KonteGlass Company, Vineland, N.J.), sonicated briefly on ice, andcentrifuged at 20,000 g for 20 minutes at 4° C. Protein concentrationwas determined using BCA protein assay (Pierce, Rockford, Ill.).Proteins were separated by electrophoresis and transferred to NuPagenitrocellulose membranes (Invitrogen, Camarillo, Calif.). Licor Odyssey™Infrared detection system (Licor, Lincoln, Nebr.) was used to assess andquantify protein expression. PI3K pathway markers were evaluated byimmunoblotting using antibodies against pAkt^(ser473) and total Akt(Invitrogen, Camarillo, Calif. and Cell Signaling, Danvers, Mass.).

Example 914 Brain Tumor In Vivo Efficacy Assay

CD-1 Nude mice (Charles River Laboratories, Hollister, Calif.) wereinoculated intracranially under stereotactic surgery with GS-2 (humanglioblastoma muliforme) cells engineered in-house to express luciferasein HBSS. Mice with confirmed brain xenografts by magnetic resonanceimaging (MRI) at four weeks post cell inoculation were dosed once dailyorally by gavage for 28 days with drug or vehicle after being separatedinto groups of similarly sized tumors. MRI (4.7T, Varian, Inc., PaloAlto, Calif.) was repeated at the end of the 28-day dosing period toassess response to treatment.

Mouse body weights were recorded at least twice weekly, and the micewere observed daily. Animal body weights were measured using anAdventurer Pro™ AV812 scale (Ohaus Corporation; Pine Brook, N.J.).Graphs were generated using GraphPad Prism, Version 5.0c. Percent weightchange was calculated using formula: individual percent weightchange=((new weight/initial weight)-1)×100. Mice whose body weight lossexceeded 20% of their starting weight were euthanized according toregulatory guidance.

The tumor volume change was modeled as linear over the two times atwhich each animal was imaged. A linear mixed effects model was fitted tothese data using the R package ‘nlme’, version 3.1-97 in R version2.12.0 (R Development Core Team 2010; R Foundation for StatisticalComputing; Vienna, Austria). A mixed effect model takes into accountrepeated measures on individual mice over time and handles theintra-mouse correlation appropriately. Linear mixed effect analysis wasalso employed to model the percent change in body weight over time.

Plasma and brain samples were collected at 2 and 8 hours postadministration of the final treatment for pharmacokinetic (PK),pharmacodynamic (PD), and/or immunohistochemical (IHC) analysis.

Example 915 In Vivo Tumor Xenograft PK/PD study

NCR nude mice (Taconic Farms, Ind.) were inoculated subcutaneously inthe right lateral thorax with 5 million U-87 MG Merchant (an in-housevariant derived from U-87 MG cells from ATCC, Manassas, Va.) cells inHBSS/Matrigel™, BD Biosciences (1:1, v/v). Mice bearing tumorxenografts >600 mm³ were dosed once with drug or vehicle after beingseparated into groups of similarly sized tumors. Plasma, subcutaneoustumor xenograft, skeletal muscle, and brain samples were collected at 1,4, 12, and 24 hours post treatment administration for PK, PD, and/or IHCanalysis.

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

We claim:
 1. A compound selected from Formula Ia:

and stereoisomers, geometric isomers, tautomers, and pharmaceuticallyacceptable salts thereof, wherein: R¹ and R² are independently selectedfrom H, ═O, C₁-C₆ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, and C₃-C₁₂carbocyclyl, where alkyl and carbocyclyl are optionally substituted withone or more groups independently selected from F, Cl, Br, I, —CH₃,—CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN, —CF₃, —CH₂OH,—CO₂H, —CONH₂, —CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂,—NHCOCH₃, —OH, ═O, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(═O)NHCH₃,—NHC(═O)NHCH₂CH₃, —NHC(═O)NHCH(CH₃)₂, —NHS(O)₂CH₃, —N(CH₃)C(═O)OC(CH₃)₃,—S(O)₂CH₃, benzyl, benzyloxy, cyclopropyl, morpholinyl,morpholinomethyl, and 4-methylpiperazin-1-yl; or two R¹ groups and thecarbon to which they are attached form a spiro, 3- to 6-memberedcarbocyclic or heterocyclic ring; or two R² groups and the carbon towhich they are attached form a spiro, 3- to 6-membered carbocyclic orheterocyclic ring; and R³ is selected from C₆-C₂₀ aryl, C₂-C₂₀heterocyclyl and C₁-C₂₀ heteroaryl, each of which are optionallysubstituted with one or more groups independently selected from F, Cl,Br, I, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN,—CF₃, —CH₂OH, —CO₂H, —CONH₂, —CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃,—N(CH₃)₂, —NHCOCH₃, —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH,—NHC(═O)NHCH₃, —NHC(═O)NHCH₂CH₃, —NHC(═O)NHCH(CH₃)₂, —NHS(O)₂CH₃,—N(CH₃)C(═O)OC(CH₃)₃, —S(O)₂CH₃, benzyl, benzyloxy, morpholinyl,morpholinomethyl, and 4-methylpiperazin-1-yl.
 2. The compound of claim 1wherein R¹ is independently selected from H, —CH₃, —CH₂CH₃, —C(CH₃)₃,—CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃, —CN, —CH₂F, —CHF₂, and —CF₃. 3.The compound of claim 1 wherein R¹ is independently selected from H,—CH₃, and cyclopropyl.
 4. The compound of claim 1 wherein R² isindependently selected from H, —CH₃, —C(CH₃)₃, —CH₂OH, —CF₃, —CH₂F, andcyclopropyl.
 5. The compound of claim 4 wherein R² are each —CH₃.
 6. Thecompound of claim 1 wherein two R² groups and the carbon to which theyare attached form ═O or cyclopropyl.
 7. The compound of claim 1 whereinR³ is phenyl substituted with one or more groups selected from F, Cl,Br, I, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CN, —CF₃, —CH₂OH, —CO₂H, —CONH₂,—CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, NHCOCH₃, —OH,—OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(═O)NHCH₃, —NHC(═O)NHCH₂CH₃,—NHS(O)₂CH₃, —N(CH₃)C(═O)OC(CH₃)₃, and —S(O)₂CH₃.
 8. The compound ofclaim 1 wherein R³ is an optionally substituted bicyclic heteroarylgroup selected from 1H-indazole, 1H-indole, indolin-2-one,1-(indolin-1-yl)ethanone, 1H-benzo[d][1,2,3]triazole,1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine,1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one,1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine,3H-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine,1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine,2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline,isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one,3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one,quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine, andpyrido[3,2-b]pyrazine.
 9. The compound of claim 1 wherein optionallysubstituted R³ is selected from:

where the wavy line indicates the site of attachment.
 10. The compoundof claim 9 wherein R³ is 1H-indazol-4-yl.
 11. The compound of claim 1wherein R³ is an optionally substituted monocyclic heteroaryl groupselected from 2-furanyl, 3-furanyl, 2-imidazolyl, 4-imidazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl,5-oxazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-pyrazinyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl, 5-pyrimidinyl,6-pyrimidinyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrrolyl, 3-pyrrolyl,2-thienyl, 3-thienyl, 5-tetrazolyl, 1-tetrazolyl, 2-tetrazolyl,2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-triazolyl, and 1-triazolyl. 12.The compound of claim 11 wherein R³ is 2-aminopyrimidin-5-yl.
 13. Thecompound of claim 1 wherein optionally substituted R³ is selected from:

where the wavy line indicates the site of attachment.
 14. A compound ofclaim 1 selected from:5-(4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine;5-[(6R)-6-methyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl]pyrimidin-2-amine;5-(7-methyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine;5-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine;5-[4-morpholino-7-(trifluoromethyl)-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl]pyrimidin-2-amine;5-(4-morpholinospiro[6H-[1,4]dioxino[2,3-d]pyrimidine-7,1′-cyclopropane]-2-yl)pyrimidin-2-amine;5-(6-cyclopropyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine;5-(7-tert-butyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine;3-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)phenol;2-(2-methoxypyrimidin-5-yl)-7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidine;5-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyridin-2-amine;N-[4-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)phenyl]acetamide;5-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)-N-methyl-pyridin-2-amine;N-[3-(7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidin-2-yl)phenyl]methanesulfonamide;2-(1H-indol-5-yl)-7,7-dimethyl-4-morpholino-6H-[1,4]dioxino[2,3-d]pyrimidine;5-[7-(fluoromethyl)-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl]pyrimidin-2-amine;[2-(2-aminopyrimidin-5-yl)-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-7-yl]methanol;and5-(7-cyclopropyl-4-morpholino-6,7-dihydro-[1,4]dioxino[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine.15. A pharmaceutical composition comprised of a compound of claim 1 anda pharmaceutically acceptable carrier, glidant, diluent, or excipient.16. The pharmaceutical composition according to claim 15, furthercomprising an additional therapeutic agent selected from achemotherapeutic agent, an anti-inflammatory agent, an immunomodulatoryagent, a neurotropic factor, an agent for treating cardiovasculardisease, an agent for treating liver disease, an anti-viral agent, anagent for treating blood disorders, an agent for treating diabetes, andan agent for treating immunodeficiency disorders.
 17. A method oftreating cancer in a patient comprised of administering to said patienta therapeutically effective amount of a compound of claim 1 wherein thecancer is breast, ovary, cervix, prostate, testis, genitourinary tract,esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin,keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma,non-small cell lung carcinoma (NSCLC), small cell carcinoma, lungadenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma andbiliary passages, kidney carcinoma, renal, pancreatic, myeloiddisorders, lymphoma, hairy cells, buccal cavity, naso-pharyngeal,pharynx, lip, tongue, mouth, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, Hodgkin's orleukemia.
 18. The method of claim 17 wherein the cancer is a braincancer.
 19. The method of claim 17 further comprising administering tothe patient an additional therapeutic agent selected from achemotherapeutic agent, an anti-angiogenesis therapeutic agent, ananti-inflammatory agent, an immunomodulatory agent, a neurotropicfactor, an agent for treating cardiovascular disease, an agent fortreating liver disease, an anti-viral agent, an agent for treating blooddisorders, an agent for treating diabetes, and an agent for treatingimmunodeficiency disorders.
 20. The method of claim 19 wherein theadditional therapeutic agent is bevacizumab.
 21. A process for making apharmaceutical composition which comprises combining a compound of claim1 with a pharmaceutically acceptable carrier.
 22. A kit for treatingbrain cancer, comprising: a) a first pharmaceutical compositioncomprising a compound of claim 1; and b) instructions for use.